IL250902B - Anti-met antibodies and compositions - Google Patents
Anti-met antibodies and compositionsInfo
- Publication number
- IL250902B IL250902B IL250902A IL25090217A IL250902B IL 250902 B IL250902 B IL 250902B IL 250902 A IL250902 A IL 250902A IL 25090217 A IL25090217 A IL 25090217A IL 250902 B IL250902 B IL 250902B
- Authority
- IL
- Israel
- Prior art keywords
- antibody
- met
- amino acid
- seq
- antigen
- Prior art date
Links
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Classifications
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Description
PCT/IB2015/002110 WO 2016/042412 ANTI-MET ANTIBODIES AND COMPOSITIONS Related Applications id="p-1" id="p-1" id="p-1" id="p-1" id="p-1"
id="p-1"
[0001]This application claims the benefit of U.S. provisional application 62/051,190, filed on September 16, 2014, which is hereby incorporated by reference in its entirety.
Sequence Listing id="p-2" id="p-2" id="p-2" id="p-2" id="p-2"
id="p-2"
[0002]The instant application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on September 15, 2015, is named 110285-0051-W01_SL.txt and is 78,490 bytes in size.
Field of the Invention id="p-3" id="p-3" id="p-3" id="p-3" id="p-3"
id="p-3"
[0003]This invention relates to anti-MET antibodies and antibody compositions and methods of using them in treating diseases and conditions related to MET.
Background of the Invention id="p-4" id="p-4" id="p-4" id="p-4" id="p-4"
id="p-4"
[0004]MET (also known as c-MET) is a receptor tyrosine kinase comprising a 50 kDa a- subunit and a 145 kDa p-subunit. The only known ligand for MET is hepatocyte growth factor (HGF), which is also known as scatter factor. Binding of HGF to MET leads to receptor dimerization and autophosphorylation of p-subunit residues Y1349 and Y1356, activating downstream signaling pathways that include the phosphoinositol 3-kinase (PI3K)- protein kinase B (Akt) pathway, the signal transducer and activator of transcription factor (STAT) pathway, the mitogen-activated protein kinase (MAPK) pathway, and the nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) pathway. This ultimately leads to increased mitogenesis, cell proliferation, cell survival, and cell motility. Dysregulation of MET or HGF activity may occur, e.g., through overexpression, gene amplification, mutation, PCT/IB2015/002110 WO 2016/042412 or alternative splicing of MET, or through HGF ligand-induced autocrine/paracrine loop signaling. Such dysregulation plays a role in many cancers by facilitating cancer invasiveness, angiogenesis, metastasis, and tumor growth, thus leading to a more aggressive cancer phenotype and a poorer prognosis. [0005]MET is also known to interact with signaling pathways involving other receptors, such as EGFR, TGF־p, and HER3, and may play a role in resistance to treatments targeting those receptors. MET inhibitors, such as anti-MET antibodies, thus may be effective in combination with other receptor inhibitors in overcoming resistant phenotypes. [0006]Current MET inhibitors include both monoclonal antibodies, which may target either MET or its ligand, HGF, and small molecule kinase inhibitors. Known antibodies targeting the MET pathway include the humanized anti-MET antibody onartuzumab (OA-5D5, OAM4558g, MetMAb); the humanized anti-HGF antibody ficlatuzumab (AV-299); the human anti-HGF antibody rilotumumab (AMG102); the humanized anti-HGF antibody TAK701; the humanized lgG4 anti-c-MET antibody LY2875358/LA480; the humanized anti-c-MET antibody ABT-700 (H224G11); and the ARGX-111 anti-c-MET antibody (36C4). Known anti- MET small molecule receptor tyrosine kinase inhibitors include tivantinib, cabozantinib, foretinib, golvatinib, and crizotinib. However, no anti-MET antibodies have been approved for therapeutic use. [0007]In view of the critical role of MET in cancer progression, there is a need for new and improved therapies that target MET.
Summary of the Invention id="p-8" id="p-8" id="p-8" id="p-8" id="p-8"
id="p-8"
[0008]The present invention is directed to novel recombinant antibodies targeting MET, as well as compositions comprising two or more of these antibodies, and use of the antibodies and compositions for treatment of cancers including non-small cell lung cancer, gastric cancer, hepatocellular carcinoma, esophageal cancer, colorectal cancer, kidney papillary cell cancer, glioblastoma, adrenocortical carcinoma, renal cell carcinoma, prostate cancer, and other cancers that express or overexpress MET or rely on MET pathway activation. Compared to currently available treatments for such cancers, including antibody treatments, it is contemplated that the antibodies of the invention provide a superior clinical response either alone or in a composition comprising two or more such antibodies. [0009]In one embodiment, the present invention provides an antibody composition comprising a first anti-MET antibody or an antigen-binding portion thereof and a second anti- MET antibody or an antigen-binding portion thereof. [0010]In some embodiments, the first anti-MET antibody competes for binding to human MET with an antibody having an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L- CDR3 comprising the amino acid sequences of SEQ ID NOs: 21, 22, 23, 24, 25, and 26, PCT/IB2015/002110 WO 2016/042412 respectively, and the second anti-MET antibody competes for binding to human MET with an antibody having an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3 comprising the amino acid sequences of SEQ ID NOs: 27, 28, 29, 30, 31, and 32, respectively. [0011]In some embodiments, the first anti-MET antibody binds to the same epitope of human MET as an antibody having an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3 comprising the amino acid sequences of SEQ ID NOs: 21, 22, 23, 24, 25, and 26, respectively, and the second anti-MET antibody binds to the same epitope of human MET as an antibody having an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDRcomprising the amino acid sequences of SEQ ID NOs: 27, 28, 29, 30, 31, and 32, respectively. In some embodiments, the first MET antibody may bind SEMA-a blade 3, and the second MET antibody may bind SEMA-a blade 2. A combination of these antibodies target both epitopes and produce surprising synergistic inhibitory effects on MET signaling pathway. We have discovered that combined targeting of these epitopes produces surprisingly high inhibitory activity on the MET signaling pathway. [0012]In some embodiments, the first anti-MET antibody comprises an H-CDR3 that comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the first anti- MET antibody comprises an H-CDR1, H-DR2, and H-CDR3 that comprise the amino acid sequences of SEQ ID NOs: 21,22, and 23, respectively. In some embodiments, the first anti-MET antibody comprises a heavy chain variable domain (VH) that is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 6 or 14. In some embodiments, the first anti-MET antibody comprises a VH comprises the amino acid sequence of SEQ ID NO: 6 or 14. In some embodiments, the first anti-MET antibody comprises a heavy chain (HC) that comprises the amino acid sequence of SEQ ID NO: 34. [0013]In some embodiments, the first anti-MET antibody comprises an L-CDR3 that comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the first anti- MET antibody comprises an L-CDR1, L-CDR2, and L-CDR3 that comprise the amino acid sequences of SEQ ID NOs: 24, 25, and 26, respectively. In some embodiments, the first anti-MET antibody comprises a light chain variable domain (VL) that is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 8 or 16. In some embodiments, the first anti-MET antibody comprises a VL that comprises the amino acid sequence of SEQ ID NO: 8 or 16. In some embodiments, the first anti-MET antibody comprises a light chain (LC) that comprises the amino acid sequence of SEQ ID NO: 33. [0014]In some embodiments, the first anti-MET antibody comprises an H-CDR3 that comprises the amino acid sequence of SEQ ID NO: 23 and an L-CDR3 that comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the first-anti-MET antibody comprises an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3 that comprise PCT/IB2015/002110 WO 2016/042412 the amino acid sequences of SEQ ID NOs: 21, 22, 23, 24, 25, and 26, respectively. In some embodiments, the first anti-MET antibody comprises a VH that is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 6 or 14 and a VL that is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 8 or 16. In some embodiments, the first anti-MET antibody comprises a VH that comprises the amino acid sequence of SEQ ID NO: 6 or 14 and a VL that comprises the amino acid sequence of SEQ ID NO: 8 or 16. In some embodiments, the first anti-MET antibody comprises a VH that comprises the amino acid sequence of SEQ ID NO: 6 and a VL that comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first anti-MET antibody comprises a VH that comprises the amino acid sequence of SEQ ID NO: 14 and a VL that comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the first anti- MET antibody comprises an HC that comprises the amino acid sequence of SEQ ID NO: and an LC that comprises the amino acid sequence of SEQ ID NO: 33. [0015]In some embodiments, the second anti-MET antibody comprises an H-CDR3 that comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the second anti-MET antibody comprises an H-CDR1, H-CDR2, and H-CDR3 that comprise the amino acid sequences of SEQ ID NOs: 27, 28, and 29, respectively. In some embodiments, the second anti-MET antibody comprises a VH that is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 10 or 18. In some embodiments, the second anti-MET antibody comprises a VH that comprises the amino acid sequence of SEQ ID NO: 10 or 18. In some embodiments, the second-anti-MET antibody comprises an HC that comprises the amino acid sequence of SEQ ID NO: 36. [0016]In some embodiments, the second anti-MET antibody comprises an L-CDR3 that comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the second anti-MET antibody comprises an L-CDR1, L-CDR2, and L-CDR3 that comprise the amino acid sequences of SEQ ID NOs: 30, 31, and 32, respectively. In some embodiments, the second anti-MET antibody comprises a VL that is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 12 or 20. In some embodiments, the second anti-MET antibody comprises the amino acid sequence of SEQ ID NO: 12 or 20. In some embodiments, the second-anti-MET antibody comprises an LC that comprises the amino acid sequence of SEQ ID NO: 35. [0017]In some embodiments, the second anti-MET antibody comprises an H-CDR3 that comprises the amino acid sequence of SEQ ID NO: 29 and an L-CDR3 that comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the second anti-MET antibody comprises an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3 that comprise the amino acid sequences of SEQ ID NOs: 27, 28, 29, 30, 31, and 32, respectively. In some embodiments, the second anti-MET antibody comprises a VH that is PCT/IB2015/002110 WO 2016/042412 at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 10 or 18 and a VL that is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO:or 20. In some embodiments, the second anti-MET antibody comprises a VH that comprises the amino acid sequence of SEQ ID NO: 10 or 18 and a VL that comprises the amino acid sequence of SEQ ID NO: 12 or 20. In some embodiments, the second anti-MET antibody comprises a VH that comprises the amino acid sequence of SEQ ID NO: 10 and a VL that comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the second anti-MET antibody comprises a VH that comprises the amino acid sequence of SEQ ID NO: 18 and a VL that comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the second anti-MET antibody comprises an HC that comprises the amino acid sequence of SEQ ID NO: 36 and an LC that comprises the amino acid sequence of SEQ ID NO: 35. [0018]The present invention also provides antibody compositions comprising any combination of the first and second anti-MET antibodies described herein. [0019]For example, in some embodiments, the first anti-MET antibody has an H-CDRand L-CDR3 comprising the amino acid sequences of SEQ ID NOs: 23 and 26, respectively, and the second anti-MET antibody has an H-CDR3 and L-CDR3 comprising the amino acid sequences of SEQ ID NOs: 29 and 32, respectively. In some embodiments, the first anti- MET antibody has an H-CDR1, H-CDR2, H-CDR3 and L-CDR1, L-CDR2, and L-CDRcomprising the amino acid sequences of SEQ ID NOs: 21, 22, 23, 24, 25, and 26, respectively, and the second anti-MET antibody has an H-CDR1, H-CDR2, H-CDR3 and L- CDR1, L-CDR2, and L-CDR3 comprising the amino acid sequences of SEQ ID NOs: 27, 28, 29, 30, 31, and 32, respectively. [0020]In some embodiments, the VH and VL of the first anti-MET antibody are at least 90% identical in sequence to the amino acid sequences of SEQ ID NOs: 6 and 8, respectively, and the VH and VL of the second anti-MET antibody are at least 90% identical in sequence to the amino acid sequences of SEQ ID NOs: 10 and 12, respectively. In some embodiments, the VH and VL of the first anti-MET antibody are at least 90% identical in sequence to the amino acid sequences of SEQ ID NOs: 14 and 16, respectively, and the VH and VL of the second anti-MET antibody are at least 90% identical in sequence to the amino acid sequences of SEQ ID NOs: 18 and 20, respectively. In some embodiments, the VH and VL of the first anti-MET antibody comprise the amino acid sequences of SEQ ID NOs: and 8, respectively, and the VH and VL of the second anti-MET antibody comprise the amino acid sequences of SEQ ID NOs: 10 and 12, respectively. In some embodiments, the VH and VL of the first anti-MET antibody comprise the amino acid sequences of SEQ ID NOs:and 16, respectively, and the VH and VL of the second anti-MET antibody comprise the amino acid sequences of SEQ ID NOs: 18 and 20, respectively. In some embodiments, the PCT/IB2015/002110 WO 2016/042412 HC and LC of the first anti-MET antibody comprise the amino acid sequences of SEQ ID NOs: 34 and 33, respectively, and the HC and LC of the second anti-MET antibody comprise the amino acid sequences of SEQ ID NOs: 36 and 35, respectively. [0021]In some embodiments of the anti-MET antibody compositions described herein, the first anti-MET antibody, the second anti-MET antibody, or both, are of isotype IgG. In certain embodiments, the first anti-MET antibody, the second anti-MET antibody, or both, are of isotype subclass lgG1. [0022]In some embodiments, at least one, at least two, or all of the anti-MET antibodies in a composition described herein have at least one property, or any combination of properties, selected from the group consisting of:• does not bind to mouse or chicken MET;• binds to an epitope of human MET comprising residues that are present on the SEMA domain;• induces degradation of MET;• binds to human MET with a KD of 1 x 10'9 M or less;• inhibits growth in vitro of at least one cell line selected from SNU5, EBC1, MKN45, Katoll, OE33, and Okajima;• inhibits MET phosphorylation;• inhibits MET downstream signaling;• inhibits primary endothelial cell proliferation in the presence or absence of HGF; and• inhibits tumor growth in vivo. [0023]In some embodiments, any of the anti-MET antibody compositions described herein has at least one property, or any combination of properties, selected from the group consisting of:• induces degradation of MET;• inhibits growth in vitro of at least one cell line selected from SNU5, EBC1, MKN45, Katoll, OE33, and Okajima;• inhibits MET phosphorylation;• inhibits MET downstream signaling;• inhibits primary endothelial cell proliferation in the presence or absence of HGF; and• inhibits tumor growth in vivo. [0024]The present invention also provides a pharmaceutical composition comprising any of the anti-MET antibody compositions described herein and a pharmaceutically acceptable excipient.
PCT/IB2015/002110 WO 2016/042412 id="p-25" id="p-25" id="p-25" id="p-25" id="p-25"
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[0025]The present invention also provides an anti-MET antibody or an antigen-binding portion thereof. In some embodiments, the antibody or portion competes for binding to human MET with an antibody whose H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L- CDR3 comprise the amino acid sequences of SEQ ID NOs: 21,22, 23, 24, 25, and 26, respectively. In some embodiments, the antibody or portion competes for binding to human MET with an antibody whose H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDRcomprise the amino acid sequences of SEQ ID NOs: 27, 28, 29, 30, 31, and 32, respectively. [0026]In some embodiments, the antibody or portion binds to the same epitope of human MET as an antibody whose H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDRcomprise the amino acid sequences of SEQ ID NOs: 21,22, 23, 24, 25, and 26, respectively. In some embodiments, the antibody or portion binds to the same epitope of human MET as an antibody whose H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L- CDR3 comprise the amino acid sequences of SEQ ID NOs: 27, 28, 29, 30, 31, and 32, respectively. [0027]In some embodiments, the antibody comprises an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 23 and/or an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 26, an H-CDR1, H-CDR2, and H-CDR3 comprising the amino acid sequences of SEQ ID NOs: 21, 22, and 23, respectively and/oran L-CDR1, L-CDR2, and L-CDRcomprising the amino acid sequences of SEQ ID NOs: 24, 25, and 26, respectively, a VH with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 6 or and/or a VL with at least 90% sequence identity to the amino acid sequence of SEQ ID NO:or 16; or a VH comprising the amino acid sequence of SEQ ID NO: 6 or 14 and/or a VL comprising the amino acid sequence of SEQ ID NO: 8 or 16. In certain embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 6 and a VL comprising the amino acid sequence of SEQ ID NO: 8. In certain embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 14 and a VL comprising the amino acid sequence of SEQ ID NO: 16. In certain embodiments, the antibody comprises an HC comprising the amino acid sequence of SEQ ID NO: 34 and an LC comprising the amino acid sequence of SEQ ID NO: 33. [0028]In some embodiments, the antibody comprises a heavy chain that comprises an H- CDR3 comprising the amino acid sequence of SEQ ID NO: 23; an H-CDR1, H-CDR2, and H-CDR3 comprising the amino acid sequences of SEQ ID NOs: 21, 22, and 23, respectively; a VH with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 6 or 14; a VH comprising the amino acid sequence of SEQ ID NO: 6 or 14; or an HC comprising the amino acid sequence of SEQ ID NO: 34; and further comprises a light chain that comprises an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 26; an L-CDR1, PCT/IB2015/002110 WO 2016/042412 L-CDR2, and L-CDR3 comprising the amino acid sequences of SEQ ID NOs: 24, 25, and 26, respectively; a VL with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 8 or 16; a VL comprising the amino acid sequence of SEQ ID NO: 8 or 16; or an LC comprising the amino acid sequence of SEQ ID NO: 33. [0029]In some embodiments, the antibody comprises an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 29 and/or an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 32; an H-CDR1, H-CDR2, and H-CDR3 comprising the amino acid sequences of SEQ ID NOs: 27, 28, and 29, respectively and/or an L-CDR1, L-CDR2, and L-CDRcomprising the amino acid sequences of SEQ ID NOs: 30, 31, and 32, respectively; a VH with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 10 or 18; and/or a VL with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: or 20; or a VH comprising the amino acid sequence of SEQ ID NO: 10 or 18 and/or a VL comprising the amino acid sequence of SEQ ID NO: 12 or 20. In certain embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 10 and a VL comprising the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 18 and a VL comprising the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the antibody comprises an HC comprising the amino acid sequence of SEQ ID NO: 36 and an LC comprising the amino acid sequence of SEQ ID NO: 35. [0030]In some embodiments, the antibody comprises a heavy chain that comprises an H- CDR3 comprising the amino acid sequence of SEQ ID NO: 29; an H-CDR1, H-CDR2, and H-CDR3 comprising the amino acid sequences of SEQ ID NOs: 27, 28, and 29, respectively; a VH with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 10 or 18; a VH comprising the amino acid sequence of SEQ ID NO: 10 or 18; or an HC comprising the amino acid sequence of SEQ ID NO: 36; and further comprises a light chain that comprises an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 32; an L-CDR1, L-CDR2, and L-CDR3 comprising the amino acid sequences of SEQ ID NOs: 30, 31, and 32, respectively; a VL with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 12 or 20; a VL comprising the amino acid sequence of SEQ ID NO: 12 or 20; or an LC comprising the amino acid sequence of SEQ ID NO: 35. [0031]In some embodiments, the antibody has an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3 comprising the amino acid sequences of SEQ ID NOs: 21,22, 23, 24, 25, and 26, respectively. [0032]In some embodiments, the antibody has an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3 comprising the amino acid sequences of SEQ ID NOs: 27, 28, 29, 30, 31, and 32, respectively.
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[0033]In some embodiments, the antibody has a heavy chain variable domain (VH) with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 6 or 14 and a light chain variable domain (VL) with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 8 or 16. [0034]In some embodiments, the antibody has a heavy chain variable domain (VH) with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 10 or 18 and a light chain variable domain (VL) with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 12 or 20. [0035]In some embodiments, the antibody has a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 6 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 8. [0036]In some embodiments, the antibody has a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 12. [0037]In some embodiments, the antibody has a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 14 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 16. [0038]In some embodiments, the antibody has a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 20. [0039]In some embodiments, the antibody has a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 34 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 33. [0040]In some embodiments, the antibody has a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 35. [0041]The invention also provides humanized versions of chimeric antibodies and antigen-binding portions described herein, particularly antibodies and antigen-binding portions with heavy and light chain amino acid sequences relating to SEQ ID NOs: 6 and 8, respectively, or SEQ ID NOs: 10 and 12, respectively. [0042]In some embodiments of the antibodies and antigen-binding portions described herein, the antibody may be of isotype IgG. In certain embodiments, the antibody is of isotype subclass lgG1. [0043]In some embodiments, the antibody has at least one property, or any combination of properties, selected from the group consisting of:• does not bind to mouse or chicken MET; PCT/IB2015/002110 WO 2016/042412 • binds to an epitope of human MET comprising residues that are present on the SEMA domain;• induces degradation of MET;• binds to human MET with a KD of 1 x 10'9 M or less;• inhibits growth in vitro of at least one cell line selected from SNU5, EBC1, MKN45, Katoll, OE33, and Okajima;• inhibits MET phosphorylation;• inhibits MET downstream signaling;• inhibits primary endothelial cell proliferation in the presence or absence of HGF; and• inhibits tumor growth in vivo. [0044]The present invention also provides a pharmaceutical composition comprising any of the anti-MET antibodies or antigen-binding portions thereof described herein and a pharmaceutically acceptable excipient. [0045]The present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes the heavy chain or an antigen-binding portion thereof, a nucleotide sequence that encodes the light chain or an antigen-binding portion thereof, or both, of an anti-MET antibody described herein. In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, or 19. [0046]The present invention also provides a vector comprising the isolated nucleic acid molecule, wherein said vector further comprises an expression control sequence. [0047]The present invention also provides a host cell comprising a nucleotide sequence that encodes the heavy chain or an antigen-binding portion thereof, a nucleotide sequence that encodes the light chain or an antigen-binding portion thereof, or both, of an anti-MET antibody described herein. In some embodiments, the host cell comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, or 19. [0048]The present invention also provides a non-human transgenic animal or plant comprising a nucleotide sequence that encodes the heavy chain or an antigen-binding portion thereof, a nucleotide sequence that encodes the light chain or an antigen-binding portion thereof, or both, of an anti-MET antibody described herein, wherein said animal or plant expresses the nucleotide sequence(s). In some embodiments, the animal or plant comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, or 19. [0049]The present invention also provides a method for producing an anti-MET antibody or antigen-binding portion thereof described herein, comprising providing the above- PCT/IB2015/002110 WO 2016/042412 described host cell, cultivating said host cell under conditions suitable for expression of the antibody or portion, and isolating the resulting antibody or portion. [0050]The present invention also provides a method for producing an anti-MET antibody composition described herein, comprising providing a first host cell capable of expressing a first anti-MET antibody or antigen-binding portion as described herein and a second host cell capable of expressing a second anti-MET antibody or antigen-binding portion as described herein, cultivating said first and second host cells under conditions suitable for expression of the antibodies or portions, and isolating the resulting antibodies or portions. In certain embodiments, the first and second host cells are cultured in a single bioreactor. In other embodiments, the first and second host cells are cultured in separate bioreactors. [0051]The present invention also provides a polyclonal cell line capable of expressing an anti-MET antibody composition, wherein said polyclonal cell line comprises a first host cell capable of expressing a first anti-MET antibody or antigen-binding portion thereof as described herein and a second host cell capable of expressing a second anti-MET antibody or antigen-binding portion thereof as described herein. [0052]The present invention also provides a bispecific binding molecule having the binding specificities of the first and second anti-MET antibodies or antigen-binding portions thereof of an anti-MET antibody composition described herein. In certain embodiments, the bispecific binding molecule comprises an antigen-binding portion of an antibody whose H- CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3 comprise the amino acid sequences of SEQ ID NOs: 21,22, 23, 24, 25, and 26, respectively; and an antigen-binding portion of an antibody whose H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDRcomprise the amino acid sequences of SEQ ID NOs: 27, 28, 29, 30, 31, and 32, respectively. [0053]The present invention also provides a method for treating a patient with a MET- mediated disorder, comprising administering to said patient an anti-MET antibody composition as described herein or a pharmaceutical composition comprising the anti-MET antibody composition. [0054]The present invention also provides a method for treating a patient with a MET- mediated disorder, comprising administering to said patient an anti-MET antibody or antigen- binding portion as described herein or a pharmaceutical composition comprising the anti- MET antibody or antigen-binding portion. [0055]The present invention also provides a method for treating a patient with cancer, comprising administering to said patient an anti-MET antibody composition as described herein or a pharmaceutical composition comprising the anti-MET antibody composition. In some embodiments, the cancer is dependent on MET activation. In certain embodiments, the cancer is non-small cell lung cancer, gastric cancer, hepatocellular carcinoma, PCT/IB2015/002110 WO 2016/042412 esophageal cancer, colorectal cancer, kidney papillary cell cancer, glioblastoma, renal cell carcinoma, prostate cancer, or adrenocortical carcinoma. [0056]The present invention also provides a method for treating a patient with cancer, comprising administering to said patient an anti-MET antibody or antigen-binding portion as described herein or a pharmaceutical composition comprising the anti-MET antibody or antigen-binding portion. Further, the present invention provides uses of an anti-MET antibody or antigen-binding portion as described herein or a pharmaceutical composition comprising the anti-MET antibody or antigen-binding portion in the manufacture of a medicament for treating cancer. Still further, the present invention provides an anti-MET antibody or antigen-binding portion as described herein or a pharmaceutical composition comprising the anti-MET antibody or antigen-binding portion for use in treating cancer. In some embodiments, the cancer is dependent on MET activation. In certain embodiments, the cancer is non-small cell lung cancer, gastric cancer, hepatocellular carcinoma, esophageal cancer, colorectal cancer, kidney papillary cell cancer, glioblastoma, renal cell carcinoma, prostate cancer, or adrenocortical carcinoma. In certain embodiments, the treatment also comprises administration of a chemotherapeutic agent, anti-neoplastic agent, anti-angiogenic agent, tyrosine kinase inhibitor, or another MET pathway inhibitor. [0057]In some of embodiments of the methods of treatment described herein, the patient is a mammal. In certain embodiments, the patient is a primate. In particular embodiments, the patient is a human.
Brief Description of the Drawings id="p-58" id="p-58" id="p-58" id="p-58" id="p-58"
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[0058]Figure 1 shows a competition matrix for thirteen MET antibodies tested against each other. An inhibition of at least 50% was used for differentiating epitope bins (gray squares). Dotted squares: Unidirectional competition. Black framed squares: Competition with identical antibody. [0059]Figure 2 shows a summary of the binding of MET antibodies to different human, mouse, chicken and chimeric MET constructs expressed on HEK293 cells. The amino acid sequence numbers (AA) refer to the human MET sequence that was exchanged to either chicken or mouse. Schematic illustrations of the different constructs are shown. The individual domains or subdomains are indicated. SP: Signal Peptide. SV5-GPI: SV5 peptide sequence followed by Glycine-Serine linker and GPI anchor. Note the illustration of the location of mutations is approximate. White squares: Human MET sequence. Grey squares: Chicken sequence. Hatched squares: Mouse sequence. Positive binding to transfected cells is indicated as +. Weak binding as (+). No binding as -. [0060]Figure 3 shows the results of a Western blot analysis of MET receptor levels in cell lines treated with negative control antibody, 9006, 9338, or 9006+9338 for 24 or 48 hours.
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[0061]Figure 4 shows the results of a Simple Western analysis of MET levels in cell lines treated with either negative control antibody, 9006+9338, or C8-H241 antibody for 24 hours. [0062]Figures 5A-5B show a Simple Western analysis of MET phosphorylation levels in cell lines treated with chimeric antibodies 9006 or 9338 or the antibody mixture 9006+93 [0063]Figures 6A-6B show a Simple Western analysis of ERK2 and AKT phosphorylation levels in cell lines treated with chimeric antibodies 9006 or 9338 or the antibody mixture 9006+9338. [0064]Figure 7A shows the number of HUVECs after treatment with chimeric antibody 9006 or 9338, the antibody mixture 9006+9338, or a control antibody. 25 pg/ml of total antibody is used (both singly and in the antibody mixture). Figure 7B depicts the results of the assay at the final timepoint, 404 hours of incubation. The data are normalized to untreated cells (100%). [0065]Figure 8A shows the number of HUVECs after treatment with chimeric antibody 9006 or 9338, the antibody mixture 9006+9338, or a control antibody, in presence of HGF at ng/ml. 25 pg/ml of total antibody is used (both singly and in the antibody mixture). Figure 8B depicts the results of the assay at the final timepoint, 404 hours of incubation/HGFstimulation. The data are normalized to untreated cells (100%). [0066]Figures 9A-9B show titration curves of the number of HUVECs after treatment with varying amounts of chimeric antibodies 9338 and 9006 (A and B, respectively). [0067]Figure 10 shows titration curves of the number of HUVECs after treatment with varying amounts of the antibody mixture 9006+9338. [0068]Figure 11 depicts the results at the final time point from Figures 9 and 10. The data are normalized to untreated cells. [0069]Figures 12A-12C show the results of a metabolic activity assay indicating the anti- proliferative effect of chimeric (left panel) or humanized (right panel) 9006, 9338, or 9006+9338 on the cell lines HCC827R1_cet#3 (12A), HCC827R1_cet#1 (12B) and MKN(12C). [0070]Figures 13A-13C show the results of a metabolic activity assay indicating the anti- proliferative effect of chimeric (left panel) or humanized (right panel) 9006, 9338, or 9006+9338 on the cell lines EBC-1 (13A), Katoll (13B), and Okajima (13C). [0071]Figure 14 shows viability results from titrations of Hu9338+Hu9006, 13-MET, 28- MET and 13-MET+28-MET antibodies on the cell lines EBC1, MKN45, SNU5 and Katoll. [0072]Figure 15 shows the effect of chimeric 9006, 9338, 9006+9338, or vehicle treatment on tumor growth of xenografts of the human non-small cell lung cancer cell line EBC-1 in mice. The grey area denotes the treatment period. [0073]Figure 16 shows the effect of treatment with chimeric 9006+9338 at four different concentrations compared to vehicle treatment on tumor growth of xenografts of the human PCT/IB2015/002110 WO 2016/042412 non-small cell lung cancer cell line EBC-1 in mice. The grey area denotes the treatment period [0074]Figure 17 shows the effect of chimeric 9006, 9338, 9006+9338, or vehicle treatment on tumor growth of xenografts of the human gastric cancer cell line MKN-45 in mice. The grey area denotes the treatment period. [0075]Figure 18 shows the effect of chimeric 9006, 9338, 9006+9338, or vehicle treatment on tumor growth of xenografts of the human gastric cancer cell line SNU5 in mice. The grey area denotes the treatment period. [0076]Figure 19 shows the effect of the chimeric antibody mixture 9006+9338 or vehicle treatment on tumor growth of xenografts of the human HCC patient derived xenograft model L11037 in mice. The grey area denotes the treatment period. [0077]Figure 20 shows the effect of 9006+9338, Hu9006+Hu9338 or vehicle treatment on tumor growth of xenografts of the human nonsmall cell lung cancer cell line EBC-1. The grey area denotes the treatment period. [0078]Figure 21 shows the effect of chimeric 9006+9338, humanized 9006+93(Hu9006+Hu9338), or vehicle treatment on tumor growth of xenografts of the human esophagogastric cancer cell line OE33. The grey area denotes the treatment period. [0079]Figure 22 shows the effect of C8-H241, Hu9006+Hu9338 or vehicle treatment on tumor growth of xenografts of the human non-small cell lung cancer cell line EBC-1 (n=mice per group). Grey areas denote the treatment periods. Dotted line denotes initiation of re-treatment of remaining mice (n=4) in the C8-H241 treated group with Hu9006+Hu9338. [0080]Figure 23 shows the effect of C8-H241, Hu9006, Hu9338, Hu9006+Hu9338 or vehicle treatment on tumor growth of xenografts of the human gastric cancer cell line Hs746T (n=8 mice per group). Grey area denotes the initial treatment period. Dotted line denotes single dose re-treatment with Hu9006+Hu9338 of remaining mice in the C8-H241, Hu9006 and Hu9338 groups. [0081]Figure 24 shows the effect of C8-H241, Hu9006+Hu9338 or vehicle treatment on tumor growth in four patient derived xenograft models (n=5 mice per group for LU0858,L1901 and LU2503; n=8 mice per group for LXFA0526). Grey area denotes the treatment period. [0082]Figure 25 shows the effect of balanced or skewed ratios of Hu9006+Hu9338 or vehicle treatment on tumor growth of xenografts of the human non-small cell lung cancer cell line EBC-1 (n=8 mice per group). Grey area denotes the treatment period. [0083]Figure 26 shows the heavy and light chain variable domain nucleotide and amino acid sequences of the chimeric 9006 antibody (SEQ ID NOs: 5-8). The CDRs (SEQ ID NOs: 21-26) are marked by arrows.
PCT/IB2015/002110 WO 2016/042412 id="p-84" id="p-84" id="p-84" id="p-84" id="p-84"
id="p-84"
[0084]Figure 27 shows the heavy and light chain variable domain nucleotide and amino acid sequences of the chimeric 9338 antibody (SEQ ID NOs: 9-12). The CDRs (SEQ ID NOs: 27-32) are marked by arrows. [0085]Figure 28 shows the heavy and light chain variable domain nucleotide and amino acid sequences of the humanized 9006 antibody (SEQ ID NOs: 13-16). The CDRs are marked by arrows (SEQ ID NOs: 21-26). [0086]Figure 29 shows the heavy and light chain variable domain nucleotide and amino acid sequences of the humanized 9338 antibody (SEQ ID NOs: 17-20). The CDRs (SEQ ID NOs: 27-32) are marked by arrows. [0087]Figure 30 shows the full-length light and heavy chain amino acid sequences of humanized antibody 9006 (SEQ ID NOs: 33 and 34, respectively) and humanized antibody 9338 (SEQ ID NOs: 35 and 36, respectively). The CDRs are marked by arrows. [0088]Figure 31 shows the structure of MET.
Detailed Description of the Invention Definitions and General Techniques id="p-89" id="p-89" id="p-89" id="p-89" id="p-89"
id="p-89"
[0089]Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art. [0090]Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. [0091]Throughout this specification and embodiments, the words "have" and "comprise," or variations such as "has," "having," "comprises," or "comprising," will be understood to PCT/IB2015/002110 WO 2016/042412 imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
Antibody-related definitions id="p-92" id="p-92" id="p-92" id="p-92" id="p-92"
id="p-92"
[0092]Unless otherwise stated, as used herein, "MET" refers to human MET (otherwise known as human c-MET). A human MET polypeptide sequence is available under NCBI Accession No. NM_000245.2, shown here as SEQ ID NO: 1. Unless otherwise specified, "human MET" refers to the amino acid sequence of SEQ ID NO: 1. Human MET also exists in a different isoform (isoform 2; SEQ ID NO: 2) in which 19 amino acids are inserted in IPT domain 3 (755-755: S -> STWWKEPLNIVSFLFCFAS (SEQ ID NO: 2)). [0093]The term "antibody" (Ab) or "immunoglobulin" (Ig), as used herein, refers to a tetramer comprising two heavy (H) chains (about 50-70 kDa) and two light (L) chains (about kDa) inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable domain (VH) and a heavy chain constant region (CH). Each light chain is composed of a light chain variable domain (VL) and a light chain constant region (CL). The VH and VL domains can be subdivided further into regions of hypervariability, termed "complementarity determining regions" (CDRs), interspersed with regions that are more conserved, termed "framework regions" (FRs). Each VH and VL is composed of three CDRs (H-CDR herein designates a CDRfrom the heavy chain; and L-CDR herein designates a CDR from the light chain) and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The assignment of amino acids to each region may be in accordance with IMGT® definitions (Lefranc et al., Dev Comp Immunol 27(1 ):55-77 (2003); or the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987 and 1991)); Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); or Chothia et al., Nature 342:878-8(1989). [0094]The term "recombinant antibody" refers to an antibody that is expressed from a cell or cell line comprising the nucleotide sequence(s) that encode the antibody, wherein said nucleotide sequence(s) are not naturally associated with the cell. [0095]The term "antibody composition" refers to a combination of two or more antibodies or antigen-binding portions thereof. An antibody composition may be monoclonal (i.e., consisting of identical antibody or antigen-binding portion molecules) or polyclonal (i.e., consisting of two or more different antibodies or antigen-binding portions reacting with the same or different epitopes on the same antigen or even on distinct, different antigens). [0096]The term "isolated protein", "isolated polypeptide" or "isolated antibody" refers to a protein, polypeptide or antibody that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) is PCT/IB2015/002110 WO 2016/042412 free of other proteins from the same species, (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be "isolated" from its naturally associated components. A protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art. [0097]As used herein, the term "germline" refers to the nucleotide and amino acid sequences of antibody genes and gene segments as they are passed from parents to offspring via germ cells. Germline sequences are distinguished from the nucleotide sequences encoding antibodies in mature B cells, which have been altered by recombination and hypermutation events during the course of B cell maturation. An antibody that "utilizes" a particular germline sequence has a nucleotide or amino acid sequence that aligns with that germline nucleotide sequence or with the amino acid sequence that it specifies more closely than with any other germline nucleotide or amino acid sequence. [0098]The term "affinity" refers to a measure of the attraction between an antigen and an antibody. The intrinsic attractiveness of the antibody for the antigen is typically expressed as the binding affinity equilibrium constant (KD) of a particular antibody-antigen interaction. An antibody is said to specifically bind to an antigen when the KD is < 1 mM, preferably < 1nM. A Kd binding affinity constant can be measured, e.g., by surface plasmon resonance (BIAcore™) or Bio-Layer Interferometry, for example using the Octet™ system. [0099]The term "koff" refers to the dissociation rate constant of a particular antibody-antigen interaction. A koff dissociation rate constant can be measured by Bio-Layer Interferometry, for example using the Octet™ system or by surface plasmon resonance (BIAcore™).. [0100]The term "epitope" as used herein refers to a portion (determinant) of an antigen that specifically binds to an antibody or a related molecule such as a bispecific binding molecule. Epitopic determinants generally consist of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and generally have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope may be "linear" or "conformational." In a linear epitope, all of the points of interaction between a protein (e.g., an antigen) and an interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction occur across amino acid residues on the protein that are separated from one another in the primary amino acid sequence. Once a desired epitope on an antigen is determined, it is possible to generate antibodies to that epitope using techniques well known in the art. Further, the generation and characterization of antibodies may elucidate information about desirable epitopes. From this information, it is PCT/IB2015/002110 WO 2016/042412 then possible to competitively screen antibodies for binding to the same or similar epitopes, e.g., by conducting competition studies to find antibodies compete for binding to the antigen. [0101]One can determine whether an antibody binds to the same epitope or cross competes for binding with an anti-MET antibody by using methods known in the art. In one embodiment, one allows the anti-MET antibody of the invention to bind to MET under saturating conditions and then measures the ability of the test antibody to bind to MET. If the test antibody is able to bind to MET at the same time as the reference anti-MET antibody, then the test antibody binds to a different epitope than the reference anti-MET antibody. However, if the test antibody is not able to bind to MET at the same time, then the test antibody binds to the same epitope, an overlapping epitope, or an epitope that is in close proximity to the epitope bound by the anti-MET antibody of the invention. This experiment can be performed using ELISA, RIA, BIACORE™, Bio-Layer Interferometry or flow cytometry. To test whether an anti-MET antibody cross-competes with another anti- MET antibody, one may use the competition method described above in two directions, i.e., determining if the known antibody blocks the test antibody and vice versa. In a preferred embodiment, the experiment is performed using Octet™. [0102]The term "chimeric antibody" refers in its broadest sense to an antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies, typically an antibody that is partially of human origin and partially of non- human origin, i.e., derived in part from a non-human animal, for example a mouse, rat or other rodent, or an avian such as a chicken. Chimeric antibodies are preferred over non- human antibodies in order to reduce the risk of a human anti-antibody response, e.g., a human anti-mouse antibody response in the case of a murine antibody. An example of a typical chimeric antibody is one in which the variable region sequences are murine while the constant region sequences are human. In the case of a chimeric antibody, the non-human parts may be subjected to further alteration in order to humanize the antibody. The chimeric antibodies described herein have murine variable domain sequences and human constant domain sequences. [0103]The term "humanize" refers to the fact that where an antibody is wholly or partially of non-human origin, for example a murine antibody obtained from immunization of mice with an antigen of interest or a chimeric antibody based on such a murine antibody, it is possible to replace certain amino acids, in particular in the framework regions and constant domains of the heavy and light chains, in order to avoid or minimize an immune response in humans. The specificity of an antibody’s interaction with a target antigen resides primarily in the amino acid residues located in the six CDRs of the heavy and light chain. The amino acid sequences within CDRs are therefore much more variable between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most PCT/IB2015/002110 WO 2016/042412 antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a specific naturally occurring antibody, or more generally any specific antibody with a given amino acid sequence, e.g., by constructing expression vectors that express CDR sequences from the specific antibody grafted into framework sequences from a different antibody. As a result, it is possible to "humanize" a non-human antibody and still substantially maintain the binding specificity and affinity of the original antibody. Although it is not possible to precisely predict the immunogenicity and thereby the human anti-antibody response of a particular antibody, non-human antibodies tend to be more immunogenic than human antibodies. Chimeric antibodies, where the foreign (usually rodent) constant regions have been replaced with sequences of human origin, have been shown to be generally less immunogenic than antibodies of fully foreign origin, and the trend in therapeutic antibodies is towards humanized or fully human antibodies. Chimeric antibodies or other antibodies of non-human origin thus can be humanized to reduce the risk of a human anti-antibody response. [0104]For chimeric antibodies, humanization typically involves modification of the framework regions of the variable region sequences. Amino acid residues that are part of complementarity determining regions (CDRs) most often will not be altered in connection with humanization, although in certain cases it may be desirable to alter individual CDR amino acid residues, for example to remove a glycosylation site, a deamidation site, an aspartate isomerization site or an undesired cysteine or methionine residue. N-linked glycosylation occurs by attachment of an oligosaccharide chain to an asparagine residue in the tripeptide sequence Asn-X-Ser or Asn-X-Thr, where X may be any amino acid except Pro. Removal of an N-glycosylation site may be achieved by mutating either the Asn or the Ser/Thr residue to a different residue, preferably by way of conservative substitution. Deamidation of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation, primarily when present in the sequence Asn-Gly, and to a lesser extent in other dipeptide sequences such as Asn-Ala. When such a deamidation site, in particular Asn-Gly, is present in a CDR sequence, it may therefore be desirable to remove the site, typically by conservative substitution to remove one of the implicated residues. [0105]Numerous methods for humanization of an antibody sequence are known in the art; see e.g., the review by Almagro & Fransson, Front Biosci. 13:1619-1633 (2008). One commonly used method is CDR grafting, which for e.g., a murine-derived chimeric antibody involves identification of human germline gene counterparts to the murine variable region genes and grafting of the murine CDR sequences into this framework. CDR grafting may be based on the Kabat CDR definitions, although a more recent publication (Magdelaine- Beuzelin et al., Crit Rev.Oncol Hematol. 64:210-225 (2007)) has suggested that the IMGT® PCT/IB2015/002110 WO 2016/042412 definition (the international ImMunoGeneTics information system®, www.imgt.org) may improve the result of the humanization (see Lefranc et al., Dev. Comp Immunol. 27:55-(2003)). In some cases, CDR grafting may reduce the binding specificity and affinity, and thus the biological activity, of a CDR-grafted non-human antibody as compared to the parenet antibody from which the CDRs are obtained. Back mutations (sometimes referred to as "framework repair") may be introduced at selected positions of the CDR-grafted antibody, typically in the framework regions, in order to reestablish the binding specificity and affinity of the parent antibody. Identification of positions for possible back mutations can be performed using information available in the literature and in antibody databases. Amino acid residues that are candidates for back mutations are typically those that are located at the surface of an antibody molecule, while residues that are buried or that have a low degree of surface exposure will not normally be altered. An alternative humanization technique to CDR grafting and back mutation is resurfacing, in which non-surface exposed residues of non- human origin are retained, while surface residues are altered to human residues. [0106]In certain cases, it may also be desirable to alter one or more CDR amino acid residues in order to improve binding affinity to the target epitope. This is known as "affinity maturation" and may optionally be performed in connection with humanization, for example in situations where humanization of an antibody leads to reduced binding specificity or affinity and it is not possible to sufficiently improve the binding specificity or affinity by back mutations alone. Various affinity maturation methods are known in the art, for example the in vitro scanning saturation mutagenesis method described by Burks et al., Proc Natl Acad Sci USA, 94:412-417 (1997), and the stepwise in vitro affinity maturation method of Wu et al. Proc Natl Acad Sci USA 95:6037-6042 (1998). [0107]The term "antigen-binding portion" of an antibody (or simply "antibody portion"), as used herein, refers to one or more portions or fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human MET, or a portion thereof). It has been shown that certain fragments of a full-length antibody can perform the antigen-binding function of the antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" include (i) a Fab fragment: a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment: a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment, which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) capable of specifically binding to an antigen. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to PCT/IB2015/002110 WO 2016/042412 form monovalent molecules (known as single chain Fv (scFv)). Also within the invention are antigen-binding molecules comprising a VH and/or a VL, In the case of a VH, the molecule may also comprise one or more of a CH1, hinge, CH2, or CH3 region. Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Other forms of single chain antibodies, such as diabodies, are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen-binding sites. [0108]Antibody portions, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, e.g., as described herein. [0109]In one embodiment, the antibody of the invention is a monoclonal antibody. As used herein, the acronym "mAb" refers to a monoclonal antibody, i.e., an antibody synthesized and secreted by an individual clonal population of cells. The clonal population can be a clonal population of immortalized cells. In some embodiments, the immortalized cells in the clonal population are hybrid cells - hybridomas - typically produced by the fusion of individual B lymphocytes from an immunized animal with individual cells from a lymphocytic tumour. [0110]The class (isotype) and subclass of anti-MET antibodies may be determined by any method known in the art. In general, the class and subclass of an antibody may be determined using antibodies that are specific for a particular class and subclass of antibody. Such antibodies are available commercially. The class and subclass can be determined by ELISA, Western Blot as well as other techniques. Alternatively, the class and subclass may be determined by sequencing all or a portion of the constant domains of the heavy and/or light chains of the antibodies, comparing their amino acid sequences to the known amino acid sequences of various classes and subclasses of immunoglobulins, and determining the class and subclass of the antibodies.
Anti-MET Antibodies id="p-111" id="p-111" id="p-111" id="p-111" id="p-111"
id="p-111"
[0111]The present invention relates to an antibody directed against human MET, or an antigen-binding portion of said antibody. The invention provides novel anti-MET antibodies 9006 and 9338 in both chimeric and humanized forms. Figures 26-30 depict the full-length (HC and LC) and variable domain (VH and VL) heavy and light chain nucleotide and amino acid sequences of these antibodies. Table 1 below provides the SEQ ID NOs of these sequences. Table 2 below provides the SEQ ID NOs for the heavy and light chain CDR PCT/IB2015/002110 WO 2016/042412 amino acid sequences of antibodies 9006 and 9338 (which are the same between the chimeric and humanized forms). The CDR sequences were assigned in accordance with IMGT® definitions.
Table 1: SEQ ID NOs for the nucleotide and amino acid sequences of the heavy and light chain variable domains of antibodies 9006 and 9338 chimeric humanizedVH VL VH VL HC LCDNA protein DNA protein DNA protein DNA protein protein protein9006 5 6 7 8 13 14 15 16 34 339338 9 10 11 12 17 18 19 20 36 35 Table 2: SEQ ID NOs for the amino acid sequences of the CDRs of antibodies 9006 and 9338 H-CDR1 H-CDR2 H-CDR3 L-CDR1 L-CDR2 L-CDR39006 21 22 23 24 25 269338 27 28 29 30 31 32 id="p-112" id="p-112" id="p-112" id="p-112" id="p-112"
id="p-112"
[0112]In certain embodiments, the invention provides:an anti-MET antibody or an antigen-binding portion thereof that competes for binding to human MET with an antibody having an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L- CDR2, and L-CDR3 that comprise the amino acid sequences of SEQ ID NOs: 21,22, 23, 24, 25, and 26, respectively;an anti-MET antibody or an antigen-binding portion thereof that binds to the same epitope of human MET as an antibody having an H-CDR1, H-CDR2, H-CDR3, L- CDR1, L-CDR2, and L-CDR3 that comprise the amino acid sequences of SEQ ID NOs: 21, 22, 23, 24, 25, and 26, respectively;an anti-MET antibody or an antigen-binding portion thereof that competes for binding to human MET with an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 6 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 8;an anti-MET antibody or an antigen-binding portion thereof that competes for binding to human MET with an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 14 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 16;an anti-MET antibody or an antigen-binding portion thereof that binds to the same epitope of human MET as an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 6 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 8; PCT/IB2015/002110 WO 2016/042412 an anti-MET antibody or an antigen-binding portion thereof that binds to the same epitope of human MET as an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 14 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 16; an anti-MET antibody or an antigen-binding portion thereof that competes for binding to human MET with an antibody having an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L- CDR2, and L-CDR3 that comprise the amino acid sequences of SEQ ID NOs: 27, 28, 29, 30, 31, and 32, respectively;an anti-MET antibody or an antigen-binding portion thereof that binds to the same epitope of human MET as an antibody having an H-CDR1, H-CDR2, H-CDR3, L- CDR1, L-CDR2, and L-CDR3 that comprise the amino acid sequences of SEQ ID NOs: 27, 28, 29, 30, 31, and 32, respectively;an anti-MET antibody or an antigen-binding portion thereof that competes for binding to human MET with an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 12;an anti-MET antibody or an antigen-binding portion thereof that competes for binding to human MET with an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 20;an anti-MET antibody or an antigen-binding portion thereof that binds to the same epitope of human MET as an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 12; an anti-MET antibody or an antigen-binding portion thereof that binds to the same epitope of human MET as an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 20; an anti-MET antibody or an antigen-binding portion thereof that competes for binding to human MET with an antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 33;an anti-MET antibody or an antigen-binding portion thereof that competes for binding to human MET with an antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 36 and a light chain comprising the amino acid sequence of SEQ ID NO: 35; PCT/IB2015/002110 WO 2016/042412 an anti-MET antibody or an antigen-binding portion thereof that binds to the same epitope of human MET as an antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 33; andan anti-MET antibody or an antigen-binding portion thereof that binds to the same epitope of human MET as an antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 36 and a light chain comprising the amino acid sequence of SEQ ID NO: 35. [0113]In one embodiment, the invention provides an anti-MET antibody or antigen-binding portion thereof having an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 23 or 29. In one embodiment, the invention provides an anti-MET antibody or antigen-binding portion thereof having an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 26 or 32. In one embodiment, the anti-MET antibody or antigen-binding portion thereof has an H- CDR3 comprising the amino acid sequence of SEQ ID NO: 23 or 29 and an L-CDRcomprising the amino acid sequence of SEQ ID NO: 26 or 32. In certain embodiments, the anti-MET antibody or antigen-binding portion thereof comprises:the H-CDR3 sequence of SEQ ID NO: 23 and the L-CDR3 sequence of SEQ ID NO: 26; orthe H-CDR3 sequence of SEQ ID NO: 29 and the L-CDR3 sequence of SEQ ID NO: 32. [0114]In one embodiment, the anti-MET antibody or antigen-binding portion thereof comprises:an H-CDR1, H-CDR2, and H-CDR3 comprising the amino acid sequences of SEQ ID NOS: 21,22, and 23, respectively; oran H-CDR1, H-CDR2, and H-CDR3 comprising the amino acid sequences of SEQ ID NOS: 27, 28, and 29, respectively. [0115]In one embodiment, the anti-MET antibody or antigen-binding portion thereof comprises:an L-CDR1, L-CDR2, and L-CDR3 comprising the amino acid sequences of SEQ ID NOS: 24, 25, and 26, respectively; oran L-CDR1, L-CDR2, and L-CDR3 comprising the amino acid sequences of SEQ ID NOS: 30, 31, and 32, respectively. [0116]In one embodiment, the anti-MET antibody or antigen-binding portion thereof comprises:an H-CDR1, H-CDR2, and H-CDR3 comprising the amino acid sequences of SEQ ID NOs: 21, 22, and 23, respectively, and an L-CDR1, L-CDR2, and L-CDR3 PCT/IB2015/002110 WO 2016/042412 comprising the amino acid sequences of SEQ ID NOS: 24, 25, and 26, respectively; oran H-CDR1, H-CDR2, and H-CDR3 comprising the amino acid sequences of SEQ ID NOs: 27, 28, and 29, respectively, and an L-CDR1, L-CDR2, and L-CDRcomprising the amino acid sequences of SEQ ID NOS: 30, 31, and 32, respectively. [0117]In one embodiment, the anti-MET antibody or antigen-binding portion thereof has a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 6, 10, 14, or 18. In one embodiment, the anti-MET antibody or antigen-binding portion thereof has a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 8, 12, 16, or 20. In one embodiment, the anti-MET antibody or antigen-binding portion thereof has a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 6, 10, 14, or 18 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 8, 12, 16, or 20. In certain embodiments, the anti-MET antibody or antigen-binding portion thereof comprises:a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 8;a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 12;a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 16; ora heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 20. [0118]In certain embodiments, the anti-MET antibody or antigen-binding portion thereof comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO: 34 and a light chain that comprises the amino acid sequence of SEQ ID NO: 33. [0119]In certain embodiments, the anti-MET antibody or antigen-binding portion thereof comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO: 36 and a light chain that comprises the amino acid sequence of SEQ ID NO: 35. [0120]In another aspect, the present invention provides a variant of an antibody or portion thereof as described above, wherein said variant differs from the antibody or portion thereof by 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions.
PCT/IB2015/002110 WO 2016/042412 id="p-121" id="p-121" id="p-121" id="p-121" id="p-121"
id="p-121"
[0121]In one embodiment, the invention provides an anti-MET antibody that comprises a heavy chain variable domain that is at least 90% identical in amino acid sequence to SEQ ID NO: 6, 10, 14, or 18, or an antigen-binding portion of said antibody. In certain embodiments, the heavy chain variable domain is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to SEQ ID NO: 6, 10, 14, or 18. In one embodiment, the invention provides an anti-MET antibody that comprises a light chain variable domain that is at least 90% identical in amino acid sequence to SEQ ID NO: 8, 12, 16, or 20, or an antigen-binding portion of said antibody. In certain embodiments, the light chain variable domain is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to SEQ ID NO: 8, 12, 16, or 20. The anti-MET antibody may also comprise any combination of the above-referenced heavy and light chain variable domains. [0122]In one embodiment, the invention provides an anti-MET antibody that comprises a heavy chain that is at least 90% identical in amino acid sequence to SEQ ID NO: 34 or 36, or an antigen-binding portion of said antibody. In certain embodiments, the heavy chain is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to SEQ ID NO: 34 or 36. In one embodiment, the invention provides an anti-MET antibody that comprises a light chain that is at least 90% identical in amino acid sequence to SEQ ID NO: 33 or 35, or an antigen-binding portion of said antibody. In certain embodiments, the light chain is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to SEQ ID NO: 33 or 35. The anti-MET antibody may also comprise any combination of the above-referenced heavy and light chain variable domains.[0123] Sequence similarity for polypeptides, which is also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as "Gap" and "Bestfit" which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA employing default or recommended parameters; a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of best overlap between the query and search sequences (Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132:185-2(2000)). Another preferred algorithm when comparing a sequence of the invention to a database containing a large number of sequences from different organisms is the computer program BLAST, especially blastp ortblastn, using default parameters. See, e.g., Altschul et PCT/IB2015/002110 WO 2016/042412 al., J. Mol. Biol. 215:403-410 (1990); Altschul et al., Nucleic Acids Res. 25:3389-402 (1997); incorporated herein by reference. [0124]The length of polypeptide sequences compared for homology will generally be at least about 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about residues. [0125]According to the invention, one type of amino acid substitution that may be made is to change one or more cysteines in the antibody, which may be chemically reactive, to another residue, such as, without limitation, alanine or serine. In one embodiment, there is a substitution of a non-canonical cysteine. The substitution can be made in a CDR or framework region of a variable domain or in the constant domain of an antibody. In some embodiments, the cysteine is canonical. [0126]Another type of amino acid substitution that may be made is to remove potential proteolytic sites in the antibody. Such sites may occur in a CDR or framework region of a variable domain or in the constant domain of an antibody. Substitution of cysteine residues and removal of proteolytic sites may decrease the risk of heterogeneity in the antibody product and thus increase its homogeneity. [0127]Another type of amino acid substitution is to eliminate asparagine-glycine pairs, which form potential deamidation sites, by altering one or both of the residues. [0128]Another type of amino acid substitution that may be made in one of the variants according to the invention is a conservative amino acid substitution. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson, Methods Mol. Biol. 243:307-31 (1994). [0129]Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine- tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.
PCT/IB2015/002110 WO 2016/042412 id="p-130" id="p-130" id="p-130" id="p-130" id="p-130"
id="p-130"
[0130]Alternatively, a conservative replacement may be defined as any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al., Science 256:1443-45 (1992). A "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix. [0131]In certain embodiments, amino acid substitutions to an antibody or antigen-binding portion of the invention are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, and (4) confer or modify other physicochemical or functional properties of such analogs, but still retain specific binding to human MET. Analogs can include various substitutions to the normally-occurring peptide sequence. For example, single or multiple amino acid substitutions, preferably conservative amino acid substitutions, may be made in the normally-occurring sequence, for example in the portion of the polypeptide outside the domain(s) forming intermolecular contacts. Amino acid substitutions can also be made in the domain(s) that form intermolecular contacts that can improve the activity of the polypeptide. A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence; e.g., a replacement amino acid should not alter the anti-parallel p-sheet that makes up the immunoglobulin binding domain that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence. In general, glycine and proline would not be used in an anti-parallel p- sheet. Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J.Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al., Nature 354:105 (1991). [0132]In another aspect of the invention, the antibody may be deimmunized to reduce its immunogenicity using the techniques described in, e.g., PCT Publications WO 98/52976 and WO 00/34317. [0133]In some embodiments, any of the anti-MET antibodies or antigen-binding portions described herein also may have at least one functional property selected from the group consisting of:does not bind to mouse or chicken MET;binds to an epitope of human MET comprising residues that are present on theSEMA domain;induces degradation of MET;binds to human MET with a KD of 1 x 10'9 M or less; PCT/IB2015/002110 WO 2016/042412 inhibits growth in vitro of at least one cell line selected from SNU5, EBC1, MKN45, Katoll, OE33, and Okajima; and inhibits tumor growth in vivo;or any combination of said functional properties. In some embodiments, binding of one or more antibodies or antigen-binding portions of the invention (and in particular an anti-MET antibody composition of the invention) to MET may inhibit the growth and proliferation of cells expressing the receptors (i.e., tumor cells). [0134]In some embodiments, any of the anti-MET antibodies or antigen-binding portions described herein may inhibit binding of HGF alpha or HGF beta to MET. In some embodiments, the antibodies or portions may inhibit binding of unprocessed HGF to MET. [0135]As used herein, the term "inhibits growth" (e.g., referring to cells) is intended to include any measurable decrease in the proliferation (increase in number of cells) or metabolism of a cell when contacted with an anti-MET antibody or antigen-binding portion or anti-MET antibody composition as compared to the growth of the same cells in the absence of the antibody or composition, e.g., inhibition of growth of a cell culture by at least about 10%, and preferably more, such as at least about 20% or 30%, more preferably at least about 40% or 50%, such as at least about 60%, 70%, 80%, 90%, 95% or 99%, or even about 100%. Growth inhibition can be determined in relevant cancer cell lines, e.g., as described in the examples below. [0136]The class of an anti-MET antibody obtained by the methods described herein may be switched with another class. In one aspect of the invention, a nucleic acid molecule encoding VL or VH is isolated using methods well-known in the art such that it does not include nucleic acid sequences encoding CL or CH. The nucleic acid molecules encoding VL or VH then are operatively linked to a nucleic acid sequence encoding a CL or CH, respectively, from a different class of immunoglobulin molecule. This may be achieved using a vector or nucleic acid molecule that comprises a CL or CH chain, as described above. For example, an anti-MET antibody that was originally IgM may be class switched to IgG.Further, the class switching may be used to convert one IgG subclass to another, e.g., from lgG1 to lgG2. A preferred method for producing an antibody of the invention with a desired isotype comprises the steps of isolating a nucleic acid molecule encoding the heavy chain of an anti-MET antibody and a nucleic acid molecule encoding the light chain of an anti-MET antibody, obtaining the variable domain of the heavy chain, ligating the variable domain of the heavy chain with the constant domain of a heavy chain of the desired isotype, expressing the light chain and the ligated heavy chain in a cell, and collecting the anti-MET antibody with the desired isotype. [0137]The anti-MET antibody of the invention can be an IgG, an IgM, an IgE, an IgA, or an IgD molecule. In one embodiment, the anti-MET antibody is an IgG molecule and is of PCT/IB2015/002110 WO 2016/042412 the lgG1, lgG2, lgG3, or lgG4 subclass. In certain embodiments, the antibody is of subclass lgG1.[0138] In certain embodiments, an antibody or antigen-binding portion thereof of the invention may be part of a larger immunoadhesion molecule, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov et al., Human Antibodies and Hybridomas 6:93-101 (1995)) and use of a cysteine residue, a marker peptide and a C- terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov et al., Mol. Immunol. 31:1047-1058 (1994)). Other examples include where one or more CDRs from an antibody are incorporated into a molecule either covalently or noncovalently to make it an immunoadhesin that specifically binds to an antigen of interest. In such embodiments, the CDR(s) may be incorporated as part of a larger polypeptide chain, may be covalently linked to another polypeptide chain, or may be incorporated noncovalently.[0139] In another embodiment, a fusion antibody or immunoadhesin may be made that comprises all or a portion of an anti-MET antibody of the invention linked to another polypeptide. In certain embodiments, only the variable domains of the anti-MET antibody are linked to the polypeptide. In certain embodiments, the VH domain of an anti-MET antibody is linked to a first polypeptide, while the VL domain of an anti-MET antibody is linked to a second polypeptide that associates with the first polypeptide in a manner such that the VH and VL domains can interact with one another to form an antigen-binding site. In another preferred embodiment, the VH domain is separated from the VL domain by a linker such that the VH and VL domains can interact with one another (e.g., single-chain antibodies). The VH-linker-VL antibody is then linked to the polypeptide of interest. In addition, fusion antibodies can be created in which two (or more) single-chain antibodies are linked to one another. This is useful if one wants to create a divalent or polyvalent antibody on a single polypeptide chain, or if one wants to create a bispecific antibody.[0140] To create a single chain antibody (scFv), the VH- and VL-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4 -Ser)3, such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH domains joined by the flexible linker. See, e.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad.Sci. USA 85:5879-5883 (1988); and McCafferty et al., Nature 348:552-554 (1990). The single chain antibody may be monovalent, if only a single VH and VL are used; bivalent, if two VH and VL are used; or polyvalent, if more than two VH and VL are used. Bispecific or polyvalent antibodies may be generated that bind specifically to human MET and to another molecule, for instance.
PCT/IB2015/002110 WO 2016/042412 id="p-141" id="p-141" id="p-141" id="p-141" id="p-141"
id="p-141"
[0141] In other embodiments, other modified antibodies may be prepared using anti-MET antibody-encoding nucleic acid molecules. For instance, "kappa bodies" (III et al., Protein Eng. 10:949-57 (1997)), "minibodies" (Martin et al., EMBO J. 13:5303-9 (1994)), "diabodies" (Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)), or"Janusins" (Traunecker et al., EMBO J. 10:3655-3659 (1991) and Traunecker et al., Int. J. Cancer (Suppl.) 7:51-(1992)) may be prepared using standard molecular biological techniques following the teachings of the specification.[0142] An anti-MET antibody or antigen-binding portion of the invention can be derivatized or linked to another molecule (e.g., another peptide or protein). In general, the antibodies or portions thereof are derivatized such that MET binding is not affected adversely by the derivatization or labeling. Accordingly, the antibodies and antibody portions of the invention are intended to include both intact and modified forms of the human anti-MET antibodies described herein. For example, an antibody or antibody portion of the invention can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detection agent, a pharmaceutical agent, and/or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).[0143] One type of derivatized antibody is produced by crosslinking two or more antibodies (of the same type or of different types, e.g., to create bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, II.[0144] An anti-MET antibody can also be derivatized with a chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrate group. These groups may be useful to improve the biological characteristics of the antibody, e.g., to increase serum half-life.[0145] An antibody according to the present invention may also be labeled. As used herein, the terms "label" or "labeled" refers to incorporation of another molecule in the antibody. In one embodiment, the label is a detectable marker, e.g., incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). In another embodiment, the label or marker can be therapeutic, e.g., a drug conjugate or toxin. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or PCT/IB2015/002110 WO 2016/042412 radionuclides (e.g., 3H, 14C, 15N,35S, 90Y, 99Tc, 1111n, 1251, 1311), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, 3-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), magnetic agents, such as gadolinium chelates, toxins such as pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance. [0146]In certain embodiments, the antibodies of the invention may be present in a neutral form (including zwitter ionic forms) or as a positively or negatively-charged species. In some embodiments, the antibodies may be complexed with a counterion to form a pharmaceutically acceptable salt. [0147]The term "pharmaceutically acceptable salt" refers to a complex comprising one or more antibodies and one or more counterions, wherein the counterions are derived from pharmaceutically acceptable inorganic and organic acids and bases. [0148]Pharmaceutically acceptable inorganic bases include metallic ions including, but are not limited to, appropriate alkali metal salts, alkaline earth metal salts and other physiological acceptable metal ions. Salts derived from inorganic bases include aluminum, ammonium, calcium, cobalt, nickel, molybdenum, vanadium, manganese, chromium, selenium, tin, copper, ferric, ferrous, lithium, magnesium, manganic or manganous salts, potassium, rubidium, sodium, and zinc, e.g., in their usual valences. [0149]Pharmaceutically acceptable acid addition salts of the antibodies of the present invention can be prepared from the following acids, including, without limitation, formic, acetic, acetamidobenzoic, adipic, ascorbic, boric, propionic, benzoic, camphoric, carbonic, cyclamic, dehydrocholic, malonic, edetic, ethylsulfuric, fendizoic, metaphosphoric, succinic, glycolic, gluconic, lactic, malic, tartaric, tannic, citric, nitric, ascorbic, glucuronic, maleic, folic, fumaric, propionic, pyruvic, aspartic, glutamic, benzoic, hydrochloric, hydrobromic, hydroiodic, lysine, isocitric, trifluoroacetic, pamoic, propionic, anthranilic, mesylic, orotic, oxalic, oxalacetic, oleic, stearic, salicylic, aminosalicylic, silicate, p-hydroxybenzoic, nicotinic, phenylacetic, mandelic, embonic, sulfonic, methanesulfonic, phosphoric, phosphonic, ethanesulfonic, ethanedisulfonic, ammonium, benzenesulfonic, pantothenic, naphthalenesulfonic, toluenesulfonic, 2-hydroxyethanesulfonic, sulfanilic, sulfuric, nitric, nitrous, sulfuric acid monomethyl ester, cyclohexylaminosulfonic, p-hydroxybutyric, glycine, PCT/IB2015/002110 WO 2016/042412 glycylglycine, glutamic, cacodylate, diaminohexanoic, camphorsulfonic, gluconic, thiocyanic, oxoglutaric, pyridoxal 5-phosphate, chlorophenoxyacetic, undecanoic, N-acetyl-L-aspartic, galactaric and galacturonic acids. [0150]Pharmaceutically acceptable organic bases include trimethylamine, diethylamine,N, N'-dibenzylethylenediamine, chloroprocaine, choline, dibenzylamine, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), procaine, cyclic amines, quaternary ammonium cations, arginine, betaine, caffeine, clemizole, 2-ethylaminoethanol, 2- diethylaminoethanol, 2-dimethylaminoethanol, ethanediamine, butylamine, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, ethylglucamine, glucamine, glucosamine, histidine, hydrabamine, imidazole, isopropylamine, methylglucamine, morpholine, piperazine, pyridine, pyridoxine, neodymium, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, tripropylamine, triethanolamine, tromethamine, methylamine, taurine, cholate, 6-amino-2-methyl-2-heptanol, 2-amino-2- methyl-1,3-propanediol, 2-amino-2-methyl-1 -propanol, aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, strontium, tricine, hydrazine, phenylcyclohexylamine, 2-(N- morpholino)ethanesulfonic acid, bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane, N- (2-acetamido)-2-aminoethanesulfonic acid, 1,4-piperazinediethanesulfonic acid, 3- morpholino-2-hydroxypropanesulfonic acid, 1,3-bis[tris(hydroxymethyl)methylamino]propane, 4-morpholinepropanesulfonic acid, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid, 2-[(2- hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid, N,N-bis(2-hydroxyethyl)-2- aminoethanesulfonic acid, 4-(N-morpholino)butanesulfonic acid, 3-(N,N-bs[2- hydroxyethyl]amino)-2-hydroxypropanesulfonic acid, 2-hydroxy-3-[tris(hydroxymethyl)methylamino]-1-propanesulfonic acid, 4-(2-hydroxyethyl)piperazine-1-(2- hydroxypropanesulfonic acid), piperazine-1,4-bis(2-hydroxypropanesulfonic acid) dihydrate, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid, //,//-bis(2-hydroxyethyl)glycine, N-(2- hydroxyethyl)piperazine-N'-(4-butanesulfonic acid), N-[tris(hydroxymethyl)methyl]-3- aminopropanesulfonic acid, N-tris(Hydroxymethyl)methyl-4-aminobutanesulfonic acid, N- (1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid, 2- (cyclohexylamino)ethanesulfonic acid, 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid, 3-(cyclohexylamino)-1-propanesulfonic acid, //-(2-acetamido)iminodiacetic acid, 4- (cyclohexylamino)-l-butanesulfonic acid, //-[tris(hydroxymethyl)methyl]glycine, 2-amino-2- (hydroxymethyl)-l,3-propanediol, and trometamol.
Anti-MET Antibody Compositions id="p-151" id="p-151" id="p-151" id="p-151" id="p-151"
id="p-151"
[0151]In one aspect, the invention provides an antibody composition comprising at least two antibodies or antigen-binding portions thereof of the invention. The term "anti-MET PCT/IB2015/002110 WO 2016/042412 antibody composition" refers to a composition comprising at least two anti-MET antibodies or antigen-binding portions thereof.[0152] In one embodiment, the antibody composition comprises a first anti-MET antibody or an antigen-binding portion thereof and a second anti-MET antibody or an antigen-binding portion thereof, wherein the first anti-MET antibody is selected from the group consisting of: an anti-MET antibody or an antigen-binding portion thereof that competes for binding to human MET with an antibody having an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L- CDR2, and L-CDR3 that comprise the amino acid sequences of SEQ ID NOs: 21,22, 23, 24, 25, and 26, respectively;an anti-MET antibody or an antigen-binding portion thereof that binds to the same epitope of human MET as an antibody having an H-CDR1, H-CDR2, H-CDR3, L- CDR1, L-CDR2, and L-CDR3 that comprise the amino acid sequences of SEQ ID NOs: 21, 22, 23, 24, 25, and 26, respectively;an anti-MET antibody or an antigen-binding portion thereof that competes for binding to human MET with an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 6 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 8;an anti-MET antibody or an antigen-binding portion thereof that competes for binding to human MET with an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 14 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 16;an anti-MET antibody or an antigen-binding portion thereof that binds to the sameepitope of human MET as an antibody having a heavy chain variable domaincomprising the amino acid sequence of SEQ ID NO: 6 and a light chain variabledomain comprising the amino acid sequence of SEQ ID NO: 8;an anti-MET antibody or an antigen-binding portion thereof that binds to the sameepitope of human MET as an antibody having a heavy chain variable domaincomprising the amino acid sequence of SEQ ID NO: 14 and a light chain variabledomain comprising the amino acid sequence of SEQ ID NO: 16;an anti-MET antibody or an antigen-binding portion thereof having an H-CDR3comprising the amino acid sequence of SEQ ID NO: 23;an anti-MET antibody or an antigen-binding portion thereof having an L-CDR3comprising the amino acid sequence of SEQ ID NO: 26;an anti-MET antibody or an antigen-binding portion thereof having an H-CDR3comprising the amino acid sequence of SEQ ID NO: 23 and an L-CDR3 comprisingthe amino acid sequence of SEQ ID NO: 26; PCT/IB2015/002110 WO 2016/042412 an anti-MET antibody or an antigen-binding portion thereof having an H-CDR1, H- CDR2, and H-CDR3 comprising the amino acid sequences of SEQ ID NOS: 21, 22, and 23, respectively;an anti-MET antibody or an antigen-binding portion thereof having an L-CDR1, L- CDR2, and L-CDR3 comprising the amino acid sequences of SEQ ID NOS: 24, 25, and 26, respectively;an anti-MET antibody or an antigen-binding portion thereof having an H-CDR1, H- CDR2, and H-CDR3 comprising the amino acid sequences of SEQ ID NOs: 21, 22, and 23, respectively, and an L-CDR1, L-CDR2, and L-CDR3 comprising the amino acid sequences of SEQ ID NOS: 24, 25, and 26, respectively; an anti-MET antibody or an antigen-binding portion thereof having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 6 or 14; an anti-MET antibody or an antigen-binding portion thereof having a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 8 or 16; an anti-MET antibody or an antigen-binding portion thereof having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 6 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 8; an anti-MET antibody or an antigen-binding portion thereof having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 14 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 16; an anti-MET antibody or an antigen-binding portion thereof having a heavy chain variable domain at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 6 and a light chain variable domain at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 8;an anti-MET antibody or an antigen-binding portion thereof having a heavy chain variable domain at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 14 and a light chain variable domain at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 16;an anti-MET antibody or an antigen-binding portion thereof having a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 33; andan anti-MET antibody or an antigen-binding portion thereof having a heavy chain at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 34 and a light chain at least 91%, 92%, 93%, 94%, PCT/IB2015/002110 WO 2016/042412 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 33;and wherein the second anti-MET antibody is selected from the group consisting of:an anti-MET antibody or an antigen-binding portion thereof that competes for binding to human MET with an antibody having an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L- CDR2, and L-CDR3 that comprise the amino acid sequences of SEQ ID NOs: 27, 28, 29, 30, 31, and 32, respectively;an anti-MET antibody or an antigen-binding portion thereof that binds to the same epitope of human MET as an antibody having an H-CDR1, H-CDR2, H-CDR3, L- CDR1, L-CDR2, and L-CDR3 that comprise the amino acid sequences of SEQ ID NOs: 27, 28, 29, 30, 31, and 32, respectively;an anti-MET antibody or an antigen-binding portion thereof that competes for binding to human MET with an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 12;an anti-MET antibody or an antigen-binding portion thereof that competes for binding to human MET with an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 20;an anti-MET antibody or an antigen-binding portion thereof that binds to the sameepitope of human MET as an antibody having a heavy chain variable domaincomprising the amino acid sequence of SEQ ID NO: 10 and a light chain variabledomain comprising the amino acid sequence of SEQ ID NO: 12;an anti-MET antibody or an antigen-binding portion thereof that binds to the sameepitope of human MET as an antibody having a heavy chain variable domaincomprising the amino acid sequence of SEQ ID NO: 18 and a light chain variabledomain comprising the amino acid sequence of SEQ ID NO: 20;an anti-MET antibody or an antigen-binding portion thereof having an H-CDR3comprising the amino acid sequence of SEQ ID NO: 29;an anti-MET antibody or an antigen-binding portion thereof having an L-CDR3comprising the amino acid sequence of SEQ ID NO: 32;an anti-MET antibody or an antigen-binding portion thereof having an H-CDR3comprising the amino acid sequence of SEQ ID NO: 29 and an L-CDR3 comprisingthe amino acid sequence of SEQ ID NO: 32; PCT/IB2015/002110 WO 2016/042412 an anti-MET antibody or an antigen-binding portion thereof having an H-CDR1, H- CDR2, and H-CDR3 comprising the amino acid sequences of SEQ ID NOS: 27, 28, and 29, respectively;an anti-MET antibody or an antigen-binding portion thereof having an L-CDR1, L- CDR2, and L-CDR3 comprising the amino acid sequences of SEQ ID NOS: 30, 31, and 32, respectively;an anti-MET antibody or an antigen-binding portion thereof having an H-CDR1, H- CDR2, and H-CDR3 comprising the amino acid sequences of SEQ ID NOs: 27, 28, and 29, respectively, and an L-CDR1, L-CDR2, and L-CDR3 comprising the amino acid sequences of SEQ ID NOS: 30, 31, and 32, respectively; an anti-MET antibody or an antigen-binding portion thereof having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 10 or 18; an anti-MET antibody or an antigen-binding portion thereof having a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 12 or 20; an anti-MET antibody or an antigen-binding portion thereof having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 12; an anti-MET antibody or an antigen-binding portion thereof having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 20; an anti-MET antibody or an antigen-binding portion thereof having a heavy chain variable domain at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 10 and a light chain variable domain at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 12;an anti-MET antibody or an antigen-binding portion thereof having a heavy chain variable domain at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 18 and a light chain variable domain at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 20;an anti-MET antibody or an antigen-binding portion thereof having a heavy chain comprising the amino acid sequence of SEQ ID NO: 36 and a light chain comprising the amino acid sequence of SEQ ID NO: 35; andan anti-MET antibody or an antigen-binding portion thereof having a heavy chain at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 36 and a light chain at least 91%, 92%, 93%, 94%, PCT/IB2015/002110 WO 2016/042412 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 35.Any combination of the above first and second anti-MET antibodies is contemplated. [0153]In one embodiment, the antibody composition comprises:an anti-MET antibody or an antigen-binding portion thereof that competes for binding to human MET with an antibody having an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L- CDR2, and L-CDR3 that comprise the amino acid sequences of SEQ ID NOs: 21,22, 23, 24, 25, and 26, respectively; andan anti-MET antibody or an antigen-binding portion thereof that competes for binding to human MET with an antibody having an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L- CDR2, and L-CDR3 that comprise the amino acid sequences of SEQ ID NOs: 27, 28, 29, 30, 31, and 32, respectively. [0154]In one embodiment, the antibody composition comprises:an anti-MET antibody or an antigen-binding portion thereof that binds to the same epitope of human MET as an antibody having an H-CDR1, H-CDR2, H-CDR3, L- CDR1, L-CDR2, and L-CDR3 that comprise the amino acid sequences of SEQ ID NOs: 21, 22, 23, 24, 25, and 26, respectively; andan anti-MET antibody or an antigen-binding portion thereof that binds to the same epitope of human MET as an antibody having an H-CDR1, H-CDR2, H-CDR3, L- CDR1, L-CDR2, and L-CDR3 that comprise the amino acid sequences of SEQ ID NOs: 27, 28, 29, 30, 31, and 32, respectively. [0155]In one embodiment, the antibody composition comprises:an anti-MET antibody or an antigen-binding portion thereof that competes for binding to human MET with an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 6 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 8; andan anti-MET antibody or an antigen-binding portion thereof that competes for binding to human MET with an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 12. [0156]In one embodiment, the antibody composition comprises:an anti-MET antibody or an antigen-binding portion thereof that competes for binding to human MET with an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 14 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 16; and PCT/IB2015/002110 WO 2016/042412 an anti-MET antibody or an antigen-binding portion thereof that competes for binding to human MET with an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 20.In one embodiment, the antibody composition comprises: an anti-MET antibody or an antigen-binding portion thereof that competes for binding to human MET with an antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 33; andan anti-MET antibody or an antigen-binding portion thereof that competes for binding to human MET with an antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 36 and a light chain comprising the amino acid sequence of SEQ ID NO: 35.In one embodiment, the antibody composition comprises: an anti-MET antibody or an antigen-binding portion thereof that binds to the same epitope of human MET as an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 6 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 8; and an anti-MET antibody or an antigen-binding portion thereof that binds to the same epitope of human MET as an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 12.In one embodiment, the antibody composition comprises: an anti-MET antibody or an antigen-binding portion thereof that binds to the same epitope of human MET as an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 14 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 16; and an anti-MET antibody or an antigen-binding portion thereof that binds to the same epitope of human MET as an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 20.In one embodiment, the antibody composition comprises: an anti-MET antibody or an antigen-binding portion thereof that binds to the same epitope of human MET as an antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 33; and id="p-157" id="p-157" id="p-157" id="p-157" id="p-157"
id="p-157"
[0157] id="p-158" id="p-158" id="p-158" id="p-158" id="p-158"
id="p-158"
[0158] id="p-159" id="p-159" id="p-159" id="p-159" id="p-159"
id="p-159"
[0159] id="p-160" id="p-160" id="p-160" id="p-160" id="p-160"
id="p-160"
[0160] PCT/IB2015/002110 WO 2016/042412 an anti-MET antibody or an antigen-binding portion thereof that binds to the same epitope of human MET as an antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 36 and a light chain comprising the amino acid sequence of SEQ ID NO: 35. [0161]In one embodiment, the antibody composition comprises:an anti-MET antibody or an antigen-binding portion thereof having an H-CDRcomprising the amino acid sequence of SEQ ID NO: 23; and an anti-MET antibody or an antigen-binding portion thereof having an H-CDRcomprising the amino acid sequence of SEQ ID NO: 29. [0162]In one embodiment, the antibody composition comprises:an anti-MET antibody or an antigen-binding portion thereof having an L-CDRcomprising the amino acid sequence of SEQ ID NO: 26; and an anti-MET antibody or an antigen-binding portion thereof having an L-CDRcomprising the amino acid sequence of SEQ ID NO: 32. [0163]In one embodiment, the antibody composition comprises:an anti-MET antibody or an antigen-binding portion thereof having an H-CDRcomprising the amino acid sequence of SEQ ID NO: 23 and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 26; andan anti-MET antibody or an antigen-binding portion thereof having an H-CDRcomprising the amino acid sequence of SEQ ID NO: 29 and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 32. [0164]In one embodiment, the antibody composition comprises:an anti-MET antibody or an antigen-binding portion thereof having an H-CDR1, H- CDR2, and H-CDR3 comprising the amino acid sequences of SEQ ID NOS: 21, 22, and 23, respectively; andan anti-MET antibody or an antigen-binding portion thereof having an H-CDR1, H- CDR2, and H-CDR3 comprising the amino acid sequences of SEQ ID NOS: 27, 28, and 29, respectively. [0165]In one embodiment, the antibody composition comprises:an anti-MET antibody or an antigen-binding portion thereof having an L-CDR1, L- CDR2, and L-CDR3 comprising the amino acid sequences of SEQ ID NOS: 24, 25, and 26, respectively; andan anti-MET antibody or an antigen-binding portion thereof having an L-CDR1, L- CDR2, and L-CDR3 comprising the amino acid sequences of SEQ ID NOS: 30, 31, and 32, respectively. [0166]In one embodiment, the antibody composition comprises: PCT/IB2015/002110 WO 2016/042412 an anti-MET antibody or an antigen-binding portion thereof having an H-CDR1, H- CDR2, and H-CDR3 comprising the amino acid sequences of SEQ ID NOs: 21, 22, and 23, respectively, and an L-CDR1, L-CDR2, and L-CDR3 comprising the amino acid sequences of SEQ ID NOS: 24, 25, and 26, respectively; and an anti-MET antibody or an antigen-binding portion thereof having an H-CDR1, H- CDR2, and H-CDR3 comprising the amino acid sequences of SEQ ID NOs: 27, 28, and 29, respectively, and an L-CDR1, L-CDR2, and L-CDR3 comprising the amino acid sequences of SEQ ID NOS: 30, 31, and 32, respectively.In one embodiment, the antibody composition comprises: an anti-MET antibody or an antigen-binding portion thereof having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 6 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 8; and an anti-MET antibody or an antigen-binding portion thereof having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 12.In one embodiment, the antibody composition comprises: an anti-MET antibody or an antigen-binding portion thereof having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 14 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 16; and an anti-MET antibody or an antigen-binding portion thereof having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 20.In one embodiment, the antibody composition comprises: an anti-MET antibody or an antigen-binding portion thereof having a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 33; andan anti-MET antibody or an antigen-binding portion thereof having a heavy chain comprising the amino acid sequence of SEQ ID NO: 36 and a light chain comprising the amino acid sequence of SEQ ID NO: 35.In one embodiment, the antibody composition comprises: an anti-MET antibody or an antigen-binding portion thereof having a heavy chain variable domain at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 6 and a light chain variable domain at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 8; and id="p-167" id="p-167" id="p-167" id="p-167" id="p-167"
id="p-167"
[0167] id="p-168" id="p-168" id="p-168" id="p-168" id="p-168"
id="p-168"
[0168] id="p-169" id="p-169" id="p-169" id="p-169" id="p-169"
id="p-169"
[0169] id="p-170" id="p-170" id="p-170" id="p-170" id="p-170"
id="p-170"
[0170] PCT/IB2015/002110 WO 2016/042412 an anti-MET antibody or an antigen-binding portion thereof having a heavy chain variable domain at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 10 and a light chain variable domain at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 12.Any combination of the above identity percentages of the first and second antibodies is contemplated. [0171]In one embodiment, the antibody composition comprises:an anti-MET antibody or an antigen-binding portion thereof having a heavy chain variable domain at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 14 and a light chain variable domain at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 16; andan anti-MET antibody or an antigen-binding portion thereof having a heavy chain variable domain at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 18 and a light chain variable domain at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 20.Any combination of the above identity percentages of the first and second antibodies is contemplated. [0172]In one embodiment, the antibody composition comprises:an anti-MET antibody or an antigen-binding portion thereof having a heavy chain at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 34 and a light chain at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 33; andan anti-MET antibody or an antigen-binding portion thereof having a heavy chain at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 36 and a light chain at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 35.Any combination of the above identity percentages of the first and second antibodies is contemplated.
Bispecific Binding Molecules id="p-173" id="p-173" id="p-173" id="p-173" id="p-173"
id="p-173"
[0173]In a further aspect, the binding specificities of any two individual antibodies disclosed herein may be combined in one bispecific binding molecule. For example, a PCT/IB2015/002110 WO 2016/042412 bispecific binding molecule may have the binding specificities of anti-MET antibodies 90and 9338. In some embodiments, the bispecific binding molecule may have the binding specificities of:an anti-MET antibody or an antigen-binding portion thereof having an H-CDR1, H- CDR2, and H-CDR3 comprising the amino acid sequences of SEQ ID NOs: 21, 22, and 23, respectively, and an L-CDR1, L-CDR2, and L-CDR3 comprising the amino acid sequences of SEQ ID NOS: 24, 25, and 26, respectively; and an anti-MET antibody or an antigen-binding portion thereof having an H-CDR1, H- CDR2, and H-CDR3 comprising the amino acid sequences of SEQ ID NOs: 27, 28, and 29, respectively, and an L-CDR1, L-CDR2, and L-CDR3 comprising the amino acid sequences of SEQ ID NOS: 30, 31, and 32, respectively. [0174]In some embodiments, the bispecific binding molecule may have the binding specificities of:an anti-MET antibody or an antigen-binding portion thereof having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 6 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 8; and an anti-MET antibody or an antigen-binding portion thereof having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 12. [0175]In some embodiments, the bispecific binding molecule may have the binding specificities of:an anti-MET antibody or an antigen-binding portion thereof having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 14 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 16; and an anti-MET antibody or an antigen-binding portion thereof having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 20. [0176]In some embodiments, the bispecific binding molecule may have the binding specificities of:an anti-MET antibody or an antigen-binding portion thereof having a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 33; andan anti-MET antibody or an antigen-binding portion thereof having a heavy chain comprising the amino acid sequence of SEQ ID NO: 36 and a light chain comprising the amino acid sequence of SEQ ID NO: 35.
PCT/IB2015/002110 WO 2016/042412 id="p-177" id="p-177" id="p-177" id="p-177" id="p-177"
id="p-177"
[0177]The bispecific binding molecule may be a dual variable domain antibody, i.e., wherein the two arms of the antibody comprise two different variable domains, or may be in the form of an antibody fragment such as a bispecific Fab fragment or a bispecific scFv.
Nucleic Acid Molecules and Vectors id="p-178" id="p-178" id="p-178" id="p-178" id="p-178"
id="p-178"
[0178]The present invention also provides nucleic acid molecules and sequences encoding anti-MET antibodies or antigen-binding portions thereof described herein. In some embodiments, different nucleic acid molecules encode the heavy chain and light chain amino acid sequences of the anti-MET antibody or an antigen-binding portion thereof. In other embodiments, the same nucleic acid molecule encodes the heavy chain and light chain amino acid sequences of the anti-MET antibody or an antigen-binding portion thereof. [0179]A reference to a nucleotide sequence encompasses its complement unless otherwise specified. Thus, a reference to a nucleic acid having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence. The term "polynucleotide" as referred to herein means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms. [0180]The invention also provides nucleotide sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to one or more of the above-recited nucleotide sequences or to a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, and 33-36. The term "percent sequence identity" in the context of nucleic acid sequences refers to the residues in two sequences that are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over a stretch of at least about nine nucleotides, usually at least about 18 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36, 48 or more nucleotides. There are a number of different algorithms known in the art which can be used to measure nucleotide sequence identity. For instance, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wisconsin. FASTA, which includes, e.g., the programs FASTA2 and FASTA3, provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132:185-219 (2000); Pearson, Methods Enzymol. 266:227-258 (1996); Pearson, J. Mol. Biol. 276:71-84 (1998); incorporated herein by reference). Unless otherwise specified, default parameters for a particular program or algorithm are used. For instance, percent sequence identity between nucleic acid sequences can be determined PCT/IB2015/002110 WO 2016/042412 using FASTA with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) or using Gap with its default parameters as provided in GCG Version 6.1, incorporated herein by reference. [0181]In one aspect, the invention provides a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, and 19. In some embodiments, the nucleic acid molecule may comprise the nucleotide sequences of SEQ ID NOs: 5 and 7, 9 and 11,13 and 15, or 17 and 19. [0182]In any of the above embodiments, the nucleic acid molecules may be isolated. [0183]In a further aspect, the present invention provides a vector suitable for expressing one of the chains of an antibody or antigen-binding portion thereof as described herein. The term "vector", as used herein, means a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In some embodiments, the vector is a plasmid, i.e., a circular double stranded piece of DNA into which additional DNA segments may be ligated. In some embodiments, the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. In some embodiments, the vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).In other embodiments, the vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). [0184]The invention provides vectors comprising nucleic acid molecules that encode the heavy chain of an anti-MET antibody of the invention or an antigen-binding portion thereof, the light chain of an anti-MET antibody of the invention or an antigen-binding portion thereof, or both the heavy and light chains of an anti-MET antibody of the invention or an antigen- binding portion thereof. The invention further provides vectors comprising nucleic acid molecules encoding fusion proteins, modified antibodies, antibody fragments, and probes thereof. [0185]A nucleic acid molecule encoding the heavy and/or light chain of an anti-MET antibody or portion thereof can be isolated from any source that produces such an antibody or portion. In various embodiments, the nucleic acid molecules are isolated from B cells that express an anti-MET antibody isolated from an animal immunized with a human MET antigen, or from an immortalized cell produced from such a B cell. Methods of isolating nucleic acids encoding an antibody are well-known in the art. mRNA may be isolated and used to produce cDNA for use in polymerase chain reaction (PCR) or cDNA cloning of PCT/IB2015/002110 WO 2016/042412 antibody genes. In certain embodiments, a nucleic acid molecule of the invention can be synthesized rather than isolated. [0186]In some embodiments, a nucleic acid molecule of the invention can comprise a nucleotide sequence encoding a VH domain from an anti-MET antibody or antigen-binding portion of the invention joined in-frame to a nucleotide sequence encoding a heavy chain constant domain from any source. Similarly, a nucleic acid molecule of the invention can comprise a nucleotide sequence encoding a VL domain from an anti-MET antibody or antigen-binding portion of the invention joined in-frame to a nucleotide sequence encoding a light chain constant domain from any source. [0187]In a further aspect of the invention, nucleic acid molecules encoding the variable domain of the heavy (VH) and/or light (VL) chains may be "converted" to full-length antibody genes. In one embodiment, nucleic acid molecules encoding the VH or VL domains are converted to full-length antibody genes by insertion into an expression vector already encoding heavy chain constant (CH) or light chain constant (CL) domains, respectively, such that the VH segment is operatively linked to the CH segment(s) within the vector, and/or the VL segment is operatively linked to the CL segment within the vector. In another embodiment, nucleic acid molecules encoding the VH and/or VL domains are converted into full-length antibody genes by linking, e.g., ligating, a nucleic acid molecule encoding a VH and/or VL domains to a nucleic acid molecule encoding a CH and/or CL domain using standard molecular biological techniques. Nucleic acid molecules encoding the full-length heavy and/or light chains may then be expressed from a cell into which they have been introduced and the anti-MET antibody isolated. [0188]The nucleic acid molecules may be used to recombinantly express large quantities of anti-MET antibodies. The nucleic acid molecules also may be used to produce chimeric antibodies, bispecific antibodies, single chain antibodies, immunoadhesins, diabodies, mutated antibodies and antibody derivatives, as described herein. [0189]In another embodiment, a nucleic acid molecule of the invention is used as a probe or PCR primer for a specific antibody sequence. For instance, the nucleic acid can be used as a probe in diagnostic methods or as a PCR primer to amplify regions of DNA that could be used, inter alia, to isolate additional nucleic acid molecules encoding variable domains of anti-MET antibodies. In some embodiments, the nucleic acid molecules are oligonucleotides. In some embodiments, the oligonucleotides are from highly variable domains of the heavy and light chains of the antibody of interest. In some embodiments, the oligonucleotides encode all or a part of one or more of the CDRs of the anti-MET antibodies or antigen-binding portions thereof of the invention as described herein. [0190]In another embodiment, the nucleic acid molecules and vectors may be used to make mutated anti-MET antibodies. The antibodies may be mutated in the variable domains PCT/IB2015/002110 WO 2016/042412 of the heavy and/or light chains, e.g., to alter a binding property of the antibody. For example, a mutation may be made in one or more of the CDR regions to increase or decrease the KD of the anti-MET antibody, to increase or decrease koff, or to alter the binding specificity of the antibody. In another embodiment, one or more mutations are made at an amino acid residue that is known to be changed compared to the germline in a monoclonal antibody of the invention. The mutations may be made in a CDR region or framework region of a variable domain, or in a constant domain. In a preferred embodiment, the mutations are made in a variable domain. In some embodiments, one or more mutations are made at an amino acid residue that is known to be changed compared to the germline in a CDR region or framework region of a variable domain of an antibody or antigen-binding portion thereof of the invention. [0191]In another embodiment, the framework region(s) are mutated so that the resulting framework region(s) have the amino acid sequence of the corresponding germline gene. A mutation may be made in a framework region or constant domain to increase the half-life of the anti-MET antibody. See, e.g., PCT Publication WO 00/09560. A mutation in a framework region or constant domain also can be made to alter the immunogenicity of the antibody, and/or to provide a site for covalent or non-covalent binding to another molecule. According to the invention, a single antibody may have mutations in any one or more of the CDRs or framework regions of the variable domain or in the constant domain. [0192]In some embodiments, the anti-MET antibodies of the invention or antigen-binding portions thereof are expressed by inserting DNAs encoding partial or full-length light and heavy chains, obtained as described above, into expression vectors such that the genes are operatively linked to necessary expression control sequences such as transcriptional and translational control sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV derived episomes, and the like. The antibody gene may be ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences may be chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors. In one embodiment, both genes are inserted into the same expression vector. The antibody genes may be inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present). [0193]A convenient vector is one that encodes a functionally complete human CH or CL immunoglobulin sequence, with appropriate restriction sites engineered so that any VH or VL PCT/IB2015/002110 WO 2016/042412 sequence can easily be inserted and expressed, as described above. In such vectors, splicing usually occurs between the splice donor site in the inserted J region and the splice acceptor site preceding the human C domain, and also at the splice regions that occur within the human CH exons. Polyadenylation and transcription termination may occur at native chromosomal sites downstream of the coding regions. The recombinant expression vector also can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene may be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the immunoglobulin chain. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein). [0194]In addition to the antibody chain genes, the recombinant expression vectors of the invention may carry regulatory sequences that control the expression of the antibody chain genes in a host cell. It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from retroviral LTRs, cytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SVpromoter/enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)), polyoma and strong mammalian promoters such as native immunoglobulin and actin promoters. For further description of viral regulatory elements, and sequences thereof, see e.g., US Patents 5,168,062, 4,510,245 and 4,968,615. Methods for expressing antibodies in plants, including a description of promoters and vectors, as well as transformation of plants, are known in the art. See, e.g., US Patent 6,517,529. Methods of expressing polypeptides in bacterial cells or fungal cells, e.g., yeast cells, are also well known in the art. [0195]In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., US Patents 4,399,216, 4,634,665 and 5,179,017). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. For example, selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection/amplification), the neo gene (for G4selection), and the glutamate synthetase gene.
PCT/IB2015/002110 WO 2016/042412 id="p-196" id="p-196" id="p-196" id="p-196" id="p-196"
id="p-196"
[0196]The term "expression control sequence" as used herein means polynucleotide sequences that are necessary to effect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. The term "control sequences" is intended to include, at a minimum, all components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
Hybridoma Methods of Producing Antibodies and Antibody Compositions of the Invention id="p-197" id="p-197" id="p-197" id="p-197" id="p-197"
id="p-197"
[0197]In certain embodiments, the invention provides methods for producing a cell line that produces a human monoclonal antibody or an antigen-binding portion thereof directed against MET, comprising (a) immunizing a non-human transgenic animal with MET, a portion of MET or a cell or tissue expressing MET; (b) allowing the transgenic animal to mount an immune response to MET; (c) isolating antibody-producing cells from the transgenic animal; (d) immortalizing the antibody-producing cells; (e) creating individual monoclonal populations of the immortalized antibody-producing cells; and (f) screening the immortalized antibody- producing cells to identify an antibody directed against MET. [0198]In another aspect, the invention provides a cell line that produces a human anti- MET antibody. In some embodiments the cell line is a hybridoma cell line. In some embodiments, the hybridomas are mouse hybridomas, as described above. In other embodiments, the hybridomas are produced in a non-human, non-mouse species such as rats, sheep, pigs, goats, cattle or horses. In another embodiment, the hybridomas are human hybridomas. [0199]In another embodiment, a transgenic animal is immunized with an MET antigen, primary cells, e.g., spleen or peripheral blood B cells, are isolated from the immunized transgenic animal and individual cells producing antibodies specific for the desired antigen are identified. Polyadenylated mRNA from each individual cell is isolated and reverse transcription polymerase chain reaction (RT-PCR) is performed using sense primers that anneal to variable domain sequences, e.g., degenerate primers that recognize most or all of the FR1 regions of human heavy and light chain variable domain genes and anti-sense PCT/IB2015/002110 WO 2016/042412 primers that anneal to constant or joining region sequences. cDNAs of the heavy and light chain variable domains are then cloned and expressed in any suitable host cell, e.g., a myeloma cell, as chimeric antibodies with respective immunoglobulin constant regions, such as the heavy chain and kor A constant domains. See Babcook et al., Proc Natl Acad Sci USA 93:7843-48 (1996). Anti-MET antibodies may then be identified and isolated as described herein.
Phage Display Libraries id="p-200" id="p-200" id="p-200" id="p-200" id="p-200"
id="p-200"
[0200]The invention provides a method for producing an anti-MET antibody or antigen- binding portion thereof comprising the steps of synthesizing a library of human antibodies on phage, screening the library with MET or an antibody-binding portion thereof, isolating phage that bind to MET, and obtaining the antibody from the phage. By way of example, one method for preparing the library of antibodies for use in phage display techniques comprises the steps of immunizing a non-human animal with MET or an antigenic portion thereof to create an immune response, extracting antibody-producing cells from the immunized animal; isolating RNA encoding heavy and light chains of antibodies of the invention from the extracted cells, reverse transcribing the RNA to produce cDNA, amplifying the cDNA using primers, and inserting the cDNA into a phage display vector such that antibodies are expressed on the phage. Recombinant anti-MET antibodies of the invention may be obtained in this way. [0201]Recombinant human anti-MET antibodies of the invention can be isolated by screening a recombinant combinatorial antibody library. Preferably the library is a scFv phage display library, generated using human VL and VH cDNAs prepared from mRNA isolated from B cells. Methods for preparing and screening such libraries are known in the art. Kits for generating phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01; and the Stratagene SurfZAP™ phage display kit, catalog no. 240612). There also are other methods and reagents that can be used in generating and screening antibody display libraries (see, e.g., U.S. Patent 5,223,409; PCT Publications. WO 92/18619, WO 91/17271, WO 92/20791, WO 92/15679, WO 93/01288, WO 92/01047, and WO 92/09690; Fuchs et al., Bio/Technology 9:1370-1372 (1991); Hay et al., Hum Antibod Hybridomas 3:81-85 (1992); Huse et al., Science 246:1275-1281 (1989); McCafferty et al., Nature 348:552-554 (1990); Griffiths et al., EMBO J 12:725-734 (1993); Hawkins et al., J Mol Biol 226:889-896 (1992); Clackson et al., Nature 352:624-628 (1991); Gram et al., Proc Natl Acad Sci USA 89:3576-3580 (1992); Garrad et al., Bio/Technology 9:1373-1377 (1991); Hoogenboom et al., NucAcid Res 19:4133-4137 (1991); and Barbas et al., Proc Natl Acad Sci USA 88:7978-7982 (1991), all incorporated herein by reference.
PCT/IB2015/002110 WO 2016/042412 id="p-202" id="p-202" id="p-202" id="p-202" id="p-202"
id="p-202"
[0202]In one embodiment, to isolate and produce human anti-MET antibodies with the desired characteristics, a human anti-MET antibody as described herein is first used to select human heavy and light chain sequences having similar binding activity toward MET, using the epitope imprinting methods described in PCT Publication WO 93/06213, incorporated herein by reference. The antibody libraries used in this method are preferably scFv libraries prepared and screened as described in PCT Publication WO 92/01047, McCafferty et al., Nature 348:552-554 (1990); and Griffiths et al., EMBO J 12:725-7(1993), all incorporated herein by reference. The scFv antibody libraries preferably are screened using human MET as the antigen. [0203]Once initial human VL and VH domains are selected, "mix and match" experiments can be performed, in which different pairs of the initially selected VL and VH segments are screened for MET binding to select preferred VL/VH pair combinations. Additionally, to further improve the quality of the antibody, the VL and VH segments of the preferred VL/VH pair(s) can be randomly mutated, preferably within the CDR3 region of VH and/or VL, in a process analogous to the in vivo somatic mutation process responsible for affinity maturation of antibodies during a natural immune response. This in vitro affinity maturation can be accomplished by amplifying VH and VL domains using PCR primers complimentary to the VH CDR3 or VL CDR3, respectively, which primers have been "spiked" with a random mixture of the four nucleotide bases at certain positions such that the resultant PCR products encode VH and VL segments into which random mutations have been introduced into the VH and/or VL CDR3 regions. These randomly mutated VH and VL segments can be re-screened for binding to MET. [0204]Following screening and isolation of an anti-MET antibody of the invention from a recombinant immunoglobulin display library, nucleic acids encoding the selected antibody can be recovered from the display package (e.g., from the phage genome) and subcloned into other expression vectors by standard recombinant DNA techniques. If desired, the nucleic acid can further be manipulated to create other antibody forms of the invention, as described herein. To express a recombinant human antibody isolated by screening of a combinatorial library, the DNA encoding the antibody is cloned into a recombinant expression vector and introduced into a mammalian host cell, as described herein.
Non-Hybridoma Host Cells and Methods of Antibody and Antibody Composition Production id="p-205" id="p-205" id="p-205" id="p-205" id="p-205"
id="p-205"
[0205]An additional aspect of the invention relates to methods for producing the antibody compositions and antibodies and antigen-binding portions thereof of the invention. One embodiment of this aspect of the invention relates to a method for producing an antibody as defined herein, comprising providing a recombinant host cell capable of expressing the antibody, cultivating said host cell under conditions suitable for expression of the antibody, PCT/IB2015/002110 WO 2016/042412 and isolating the resulting antibody. Antibodies produced by such expression in such recombinant host cells are referred to herein as "recombinant antibodies". The invention also provides progeny cells of such host cells, and antibodies produced by same. [0206]The term "recombinant host cell" (or simply "host cell"), as used herein, means a cell into which a recombinant expression vector has been introduced. The invention provides host cells that may comprise, e.g., a vector according to the invention described above. The invention also provides host cells that comprise, e.g., a nucleotide sequence encoding the heavy chain or an antigen-binding portion thereof, a nucleotide sequence encoding the light chain or an antigen-binding portion thereof, or both, of an anti-MET antibody or antigen-binding portion thereof of the invention. It should be understood that "recombinant host cell" and "host cell" mean not only the particular subject cell but also the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. [0207]Nucleic acid molecules encoding anti-MET antibodies and vectors comprising these nucleic acid molecules can be used for transfection of a suitable mammalian, plant, bacterial or yeast host cell. Transformation can be by any known method for introducing polynucleotides into a host cell. Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei. In addition, nucleic acid molecules may be introduced into mammalian cells by viral vectors. Methods of transforming cells are well known in the art. See, e.g., US Patents 4,399,216, 4,912,040, 4,740,461, and 4,959,455. Methods of transforming plant cells are well known in the art, including, e.g., Agrobacterium-mediated transformation, biolistic transformation, direct injection, electroporation and viral transformation. Methods of transforming bacterial and yeast cells are also well known in the art. [0208]Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, 293 Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and a number of other cell lines. Cell lines of particular preference are selected through determining which cell lines have high expression levels. Other cell lines that may be used are insect cell lines, such as Sf9 or Sf21 cells.
PCT/IB2015/002110 WO 2016/042412 When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods. Plant host cells include, e.g., Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, etc. Bacterial host cells include E. coli and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae and Pichia pastoris. [0209]Further, expression of antibodies of the invention or antigen-binding portions thereof from production cell lines can be enhanced using a number of known techniques.For example, the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions. The GS system is discussed in whole or part in connection with EP patents 0 216 846, 0 256 055, 0 323 9and 0 338 841. [0210]It is likely that antibodies expressed by different cell lines or in transgenic animals will have different glycosylation patterns from each other. However, all antibodies encoded by the nucleic acid molecules provided herein, or comprising the amino acid sequences provided herein are part of the instant invention, regardless of the glycosylation state of the antibodies, and more generally, regardless of the presence or absence of post-translational modification(s). [0211]In a further embodiment, the invention relates to a method for producing an antibody composition comprising at least two anti-MET antibodies, the method comprising: providing at least first and second host cells, wherein the first host cell is capable of expressing a first anti-MET antibody of the invention and the second host cell is capable of expressing a second anti-MET antibody of the invention; cultivating the first and second host cells under conditions suitable for expression of the anti-MET antibodies; and isolating the resulting antibodies. [0212]An antibody or antigen-binding portion thereof or antibody composition of the present invention may be produced by methods generally known in the art for production of recombinant monoclonal or polyclonal antibodies. Thus, in the case of production of a single antibody of the invention, any method known in the art for production of recombinant monoclonal antibodies may be used. For production of an antibody composition of the invention comprising a mixture of antibodies, the individual antibodies may be produced separately, i.e., each antibody being produced in a separate bioreactor, or the individual antibodies may be produced together in single bioreactor. If the antibody composition is produced in more than one bioreactor, the purified antibody composition can be obtained by PCT/IB2015/002110 WO 2016/042412 pooling the antibodies obtained from individually purified supernatants from each bioreactor. Various approaches for production of a polyclonal antibody composition in multiple bioreactors, where the cell lines or antibody preparations are combined at a later point upstream or prior to or during downstream processing, are described in WO 2009/1298(incorporated herein by reference). [0213]In the case of producing individual antibodies in a single bioreactor, this may be performed, e.g., as described in WO 2004/061104 or WO 2008/145133 (both of which are incorporated herein by reference). The method described in WO 2004/061104 is based on site-specific integration of the antibody coding sequence into the genome of the individual host cells, while the method of WO 2008/145133 involves an alternative approach using random integration to produce antibodies in a single bioreactor. [0214]Further information regarding methods suitable for preparing the antibodies and compositions of the invention may be found in WO 2012/059857 (incorporated herein by reference).
Transgenic Animals and Plants id="p-215" id="p-215" id="p-215" id="p-215" id="p-215"
id="p-215"
[0215]Anti-MET antibodies and antigen-binding portions thereof of the invention also can be produced transgenically through the generation of a mammal or plant that is transgenic for the immunoglobulin heavy and light chain sequences of interest and production of the antibody in a recoverable form therefrom. In connection with transgenic production in mammals, anti-MET antibodies and portions can be produced in, and recovered from, the milk of goats, cows, or other mammals. See, e.g., US patents 5,827,690, 5,756,687, 5,750,172, and 5,741,957. In some embodiments, non-human transgenic animals that comprise human immunoglobulin loci are immunized with human MET or an immunogenic portion thereof, as described above. Methods for making antibodies in plants are described, e.g., in US patents 6,046,037 and 5,959,177. [0216]In some embodiments, non-human transgenic animals or plants are produced by introducing one or more nucleic acid molecules encoding an anti-MET antibody or antigen- binding portion thereof of the invention (e.g., any of the above-described nucleic acid molecules encoding an anti-MET antibody or antigen-binding portion thereof) into the animal or plant by standard transgenic techniques. See, e.g., US Patent 6,417,429. The transgenic cells used for making the transgenic animal can be embryonic stem cells or somatic cells or a fertilized egg. The transgenic non-human organisms can be chimeric, nonchimeric heterozygotes, and nonchimeric homozygotes. See, e.g., Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual 2nd ed., Cold Spring Harbor Press (1999); Jackson et al., Mouse Genetics and Transgenics: A Practical Approach, Oxford University Press (2000); and Pinkert, Transgenic Animal Technology: A Laboratory Handbook, Academic PCT/IB2015/002110 WO 2016/042412 Press (1999). In some embodiments, the transgenic non-human animals have a targeted disruption and replacement by a targeting construct that encodes a heavy chain and/or a light chain of interest. The non-human transgenic animals or plants may comprise, e.g., a nucleotide sequence encoding the heavy chain or an antigen-binding portion thereof, a nucleotide sequence encoding the light chain or an antigen-binding portion thereof, or both, of an anti-MET antibody of the invention. In a preferred embodiment, the transgenic animals comprise and express nucleic acid molecules encoding heavy and light chains, or antigen- binding portions thereof, that specifically bind to human MET. The anti-MET antibodies or portions may be made in any transgenic animal. In a preferred embodiment, the non-human animals are mice, rats, sheep, pigs, goats, cattle or horses. The non-human transgenic animal may express said encoded polypeptides in, e.g., blood, milk, urine, saliva, tears, mucus and other bodily fluids.
Pharmaceutical Compositions id="p-217" id="p-217" id="p-217" id="p-217" id="p-217"
id="p-217"
[0217]Another aspect of the invention is a pharmaceutical composition comprising as an active ingredient (or as the sole active ingredient) an anti-MET antibody or antigen-binding portion thereof or anti-MET antibody composition of the invention. The pharmaceutical composition may comprise any anti-MET antibody composition or antibody or antigen- binding portion thereof as described herein. In some embodiments, the compositions are intended for amelioration, prevention, and/or treatment of a MET-mediated disorder (e.g., a disorder characterized by overexpression of MET) and/or cancer. In certain embodiments, the compositions are intended for amelioration, prevention, and/or treatment of non-small cell lung cancer, gastric cancer, hepatocellular carcinoma, esophageal cancer, colorectal cancer, kidney papillary cell cancer, glioblastoma, renal cell carcinoma, prostate cancer, and/or adrenocortical carcinoma. [0218]Generally, the antibodies of the invention or antigen-binding portions thereof are suitable to be administered as a formulation in association with one or more pharmaceutically acceptable excipient(s). The term "excipient" is used herein to describe any ingredient other than the compound(s) of the invention. The choice of excipient(s) will to a large extent depend on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. As used herein, "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in PCT/IB2015/002110 WO 2016/042412 the composition. Additional examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody. [0219]Pharmaceutical compositions of the present invention and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation may be found, for example, in Remington’s Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995). Pharmaceutical compositions are preferably manufactured under GMP (good manufacturing practices) conditions. [0220]A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage. [0221]Any method for administering peptides, proteins or antibodies accepted in the art may suitably be employed for the antibodies and antigen-binding portions of the invention. [0222]The pharmaceutical compositions of the invention are typically suitable for parenteral administration. As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue, thus generally resulting in the direct administration into the blood stream, into muscle, or into an internal organ. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intrasynovial injection or infusions; and kidney dialytic infusion techniques. Regional perfusion is also contemplated. Preferred embodiments include the intravenous and the subcutaneous routes. [0223]Formulations of a pharmaceutical composition suitable for parenteral administration typically comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Formulations for parenteral PCT/IB2015/002110 WO 2016/042412 administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. Parenteral formulations also include aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water. Exemplary parenteral administration forms include solutions or suspensions in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, or in a liposomal preparation. Formulations for parenteral administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release. [0224]For example, in one aspect, sterile injectable solutions can be prepared by incorporating the anti-MET antibody or antigen-binding portion thereof or anti-MET antibody composition in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin, and/or by using modified-release coatings (e.g., slow-release coatings). [0225]The antibodies of the invention can also be administered intranasally or by inhalation, typically in the form of a dry powder (either alone, as a mixture, or as a mixed component particle, for example, mixed with a suitable pharmaceutically acceptable excipient) from a dry powder inhaler, as an aerosol spray from a pressurised container, PCT/IB2015/002110 WO 2016/042412 pump, spray, atomiser (preferably an atomiser using electrohydrodynamics to produce a fine mist), or nebuliser, with or without the use of a suitable propellant, or as nasal drops. [0226]The pressurised container, pump, spray, atomizer, or nebuliser generally contains a solution or suspension of an antibody of the invention comprising, for example, a suitable agent for dispersing, solubilising, or extending release of the active, a propellant(s) as solvent. [0227]Prior to use in a dry powder or suspension formulation, the drug product is generally micronised to a size suitable for delivery by inhalation (typically less than microns). This may be achieved by any appropriate comminuting method, such as spiral jet milling, fluid bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenisation, or spray drying. [0228]Capsules, blisters and cartridges for use in an inhaler or insufflator may be formulated to contain a powder mix of the compound of the invention, a suitable powder base and a performance modifier. [0229]A suitable solution formulation for use in an atomiser using electrohydrodynamics to produce a fine mist may contain a suitable dose of the antibody of the invention per actuation and the actuation volume may for example vary from 1 pL to 100 pL. [0230]Suitable flavours, such as menthol and levomenthol, or sweeteners, such as saccharin or saccharin sodium, may be added to those formulations of the invention intended for inhaled/intranasal administration. [0231]Formulations for inhaled/intranasal administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release. [0232]In the case of dry powder inhalers and aerosols, the dosage unit is determined by means of a valve which delivers a metered amount. Units in accordance with the invention are typically arranged to administer a metered dose or "puff’ of an antibody of the invention. The overall daily dose will typically be administered in a single dose or, more usually, as divided doses throughout the day. [0233]The antibodies and antibody portions of the invention may also be formulated for an oral route administration. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, and/or buccal, lingual, or sublingual administration by which the compound enters the blood stream directly from the mouth. [0234]Formulations suitable for oral administration include solid, semi-solid and liquid systems such as tablets; soft or hard capsules containing multi- or nano-particulates, liquids, or powders; lozenges (including liquid-filled); chews; gels; fast dispersing dosage forms; films; ovules; sprays; and buccal/mucoadhesive patches.
PCT/IB2015/002110 WO 2016/042412 id="p-235" id="p-235" id="p-235" id="p-235" id="p-235"
id="p-235"
[0235]Liquid formulations include suspensions, solutions, syrups and elixirs. Such formulations may be employed as fillers in soft or hard capsules (made, for example, from gelatin or hydroxypropylmethylcellulose) and typically comprise a carrier, for example, water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifying agents and/or suspending agents. Liquid formulations may also be prepared by the reconstitution of a solid, for example, from a sachet.
Immunoconjugates id="p-236" id="p-236" id="p-236" id="p-236" id="p-236"
id="p-236"
[0236]Another option for therapeutic use of the antibody compositions and antibodies and antigen-binding portions thereof of the invention is in the form of immunoconjugates, i.e.,antibodies or antigen-binding portions conjugated to one or more agents such as anti- cancer agents. Compositions of the invention comprising two or more anti-MET antibodies may contain a single antibody in the form of an immunoconjugate, or they may contain two or more antibodies in the form of an immunoconjugate. [0237]Various types of anti-cancer agents may be conjugated to the antibodies of the invention, including cytotoxic agents (e.g., conventional chemotherapy agents and other small molecule anti-cancer drugs), cytokines (in which case the conjugate may be termed an "immunocytokine"), toxins (in which case the conjugate may be termed an "immunotoxin") and radionuclides. A few immunoconjugates have already been approved for clinical use. These include Zevalin® (a murine anti-CD20 antibody conjugated to 90Y), Bexxar® (a murine anti-CD20 antibody conjugated to 131I) and Mylotarg® (a humanized anti-CD33 antibody conjugated to calicheamicin). Other immunoconjugates that have been tested in clinical trials include antibodies conjugated to, e.g., doxorubicin or a maytansinoid compound. Immunotoxins that have been tested in clinical trials include several antibodies conjugated to a truncated Pseudomonas exotoxin A. An immunocytokine comprising a humanized EpCAM antibody conjugated to IL-2 has also been tested. [0238]In the case of antibodies of the invention conjugated to cytotoxic agents, these may belong, e.g., to any of the major classes of chemotherapy drugs, including alkylating agents (e.g., carboplatin, cisplatin, oxaliplatin), antimetabolites (e.g., methotrexate, capecitabine, gemcitabine), anthracyclines (e.g., bleomycin, doxorubicin, mitomycin-C) and plant alkaloids (e.g., taxanes such as docetaxel and paclitaxel, and vinca alkaloids such as vinblastine, vincristine and vinorelbine). Since the use of immunoconjugates specifically directs the anti- cancer agent to the tumors, immunoconjugates based on the antibodies of the invention may advantageously be based on highly cytotoxic agents such as calicheamicin or maytansine derivatives, or on toxins such as bacterial toxins (e.g., Pseudomonas exotoxin A, diphtheria toxin) or plant toxins (e.g., ricin).
PCT/IB2015/002110 WO 2016/042412 id="p-239" id="p-239" id="p-239" id="p-239" id="p-239"
id="p-239"
[0239] The conjugated anti-cancer agent in an immunoconjugate is generally linked to the antibody by means of a labile linker that is relatively stable in serum but which allows release of the agent when the immunoconjugate is internalized into the target cell. Suitable linkers include, for example, chemical linkers that are stable at neutral pH in serum but are subjected to acid hydrolysis in the mildly acidic conditions within the lysosomes subsequent to internalization, disulfide linkers that are cleaved by intracellular thiols, and peptide linkers that are stable in serum but which are subjected to enzymatic cleavage in intracellular compartments.[0240] Various conjugation arrangements can be envisioned in compositions containing two or more antibodies of the invention. For example, with two antibodies it would be possible to conjugate the antibodies to two or more different anti-cancer drugs or to conjugate one antibody to a prodrug which is activated by an agent such as an enzyme conjugated to the other antibody. The general concept of antibody-directed enzyme prodrug therapy (ADEPT) has been described for monoclonal antibodies, where a prodrug is activated by an enzyme targeted to the tumor by an mAB-enzyme conjugate, but the present invention may provide an opportunity for tailoring this approach to particular conditions. It may thus be possible to specifically increase tumor cell killing while sparing or reducing damage to normal tissues.[0241] For further information on anti-cancer immunoconjugates, see Wu et al., Nature Biotechnology 23(9): 1137-1146 (2005); Schrama et al., Nature Reviews/Drug Discovery 5:147-159 (2006); and Rohrer, Chimica Oggi/Chemistry Today 27(5):56-60 (2009).
Therapeutic uses of antibodies and compositions of the invention id="p-242" id="p-242" id="p-242" id="p-242" id="p-242"
id="p-242"
[0242] In one aspect, the anti-MET antibodies and antigen-binding portions thereof and anti-MET compositions of the invention are used in the treatment of a MET-mediated disorder. In some embodiments, the MET-mediated disorder is a condition characterized by overexpression of MET. In certain embodiments, the pharmaceutical composition is for use in the treatment of cancer, e.g., non-small cell lung cancer, gastric cancer, hepatocellular carcinoma, esophageal cancer, colorectal cancer, kidney papillary cell cancer, glioblastoma, adrenocortical carcinoma, renal cell carcinoma, prostate cancer, and other cancers that express or overexpress MET or rely on MET pathway activation.[0243] In some aspects, the antibodies or antibody compositions are used to treat a disorder, such as a cancer, characterized by abnormal MET overactivity. In some embodiments, the abnormal overactivity stems from gene amplification, protein overexpression, a MET activating gene mutation (e.g., a point mutation or abnormal gene splicing event), or HGF overexpression.
PCT/IB2015/002110 WO 2016/042412 id="p-244" id="p-244" id="p-244" id="p-244" id="p-244"
id="p-244"
[0244] In certain aspects, the anti-MET antibodies and antigen-binding portions thereof and anti-MET compositions of the invention may be used to treat a patient who is resistant to treatment with an agent targeting a different tyrosine kinase receptor. In some embodiments, the patient is resistant to treatment with an ErbB kinase inhibitor. In certain embodiments, the ErbB kinase inhibitor targets EGFR, ErbB2, ErbB3, or ErbB4. In a particular embodiment, the ErbB kinase inhibitor targets EGFR. In another embodiment, the ErbB kinase inhibitor targets HER3. The ErbB kinase inhibitor may be selected from, e.g., gefitinib, erlotinib, cetuximab, pantinumumab, trastuzumab, or any combination thereof. [0245] " Treat", "treating" and "treatment" refer to a method of alleviating or abrogating abiological disorder and/or at least one of its attendant symptoms. As used herein, to "alleviate" a disease, disorder or condition means reducing the severity and/or occurrence frequency of the symptoms of the disease, disorder, or condition. Further, references herein to "treatment" include references to curative, palliative and prophylactic treatment.[0246] In one aspect, the subject of treatment, or patient, is a mammal, preferably a human subject. Said subject may be either male or female, of any age.[0247] "Therapeutically effective amount" refers to that amount of the therapeutic agent being administered which will relieve to some extent one or more of the symptoms of the disorder being treated.[0248] The ratio between the individual antibodies in a therapeutic composition of the invention, or in the case of individual antibodies of the invention being administered simultaneously, sequentially or separately, will often be such that the antibodies are administered in equal amounts, but this need not necessarily be the case. Thus, a composition of the invention comprising two anti-MET antibodies or antigen-binding portions thereof will often contain them in approximately a 1:1 ratio, but depending on the characteristics of the individual antibodies, it may be desirable to use non-equal amounts of the antibodies or portions. For example, the ratio of one antibody or portion relative to another antibody or portion in a two-antibody composition may be, e.g., between 5 and 95%, between 10 and 90%, between 20 and 80%, between 30 and 70%, between 40 and 60%, or between 45 and 55%.[0249] The antibody compositions or antibodies or antigen-binding portions thereof of the invention may be administered alone or in combination with one or more other drugs or antibodies (or as any combination thereof). The pharmaceutical compositions, methods and uses of the invention thus also encompass embodiments of combinations (co-administration) with other active agents, as detailed below.[0250] As used herein, the terms "co-administration", "co-administered" and "in combination with," referring to the antibody compositions and antibodies and antigen-binding PCT/IB2015/002110 WO 2016/042412 portions thereof with one or more other therapeutic agents, is intended to mean, and does refer to and include the following:simultaneous administration of such combination of antibody composition / antibody / antigen-binding portion of the invention and therapeutic agent(s) to a patient in need of treatment, when such components are formulated together into a single dosage form which releases said components at substantially the same time to said patient,substantially simultaneous administration of such combination of antibody composition / antibody / antigen-binding portion of the invention and therapeutic agent(s) to a patient in need of treatment, when such components are formulated apart from each other into separate dosage forms which are taken at substantially the same time by said patient, whereupon said components are released at substantially the same time to said patient, sequential administration of such combination of antibody composition / antibody / antigen-binding portion of the invention and therapeutic agent(s) to a patient in need of treatment, when such components are formulated apart from each other into separate dosage forms which are taken at consecutive times by said patient with a significant time interval between each administration, whereupon said components are released at substantially different times to said patient; andsequential administration of such combination of antibody composition / antibody / antigen-binding portion of the invention and therapeutic agent(s) to a patient in need of treatment, when such components are formulated together into a single dosage form which releases said components in a controlled manner whereupon they are concurrently, consecutively, and/or overlappingly released at the same and/or different times to said patient, where each part may be administered by either the same or a different route.[0251] The antibody compositions and antibodies and antigen-binding portions thereof of the invention may be administered without additional therapeutic treatments, i.e., as a stand- alone therapy. Alternatively, treatment with the antibody compositions and antibodies and antigen-binding portions thereof of the invention may include at least one additional therapeutic treatment (combination therapy). In some embodiments, the antibody composition or antibody or antigen-binding portion thereof may be co-administered or formulated with another medication/drug for the treatment of cancer. The additional therapeutic treatment may comprise, e.g., a chemotherapeutic, anti-neoplastic, or anti- angiogenic agent, a different anti-cancer antibody, and/or radiation therapy.[0252] By combining the antibody compositions, antibodies, or antigen-binding portions of the invention with agents known to induce terminal differentiation of cancer cells, the effect may be improved further. Such compounds may, for example, be selected from the group consisting of retinoic acid, trans-retinoic acids, cis-retinoic acids, phenylbutyrate, nerve growth factor, dimethyl sulfoxide, active form vitamin D3, peroxisome proliferator-activated PCT/IB2015/002110 WO 2016/042412 receptor gamma, 12-O-tetradecanoylphorbol 13-acetate, hexamethylene-bis-acetamide, transforming growth factor-beta, butyric acid, cyclic AMP, and vesnarinone. In some embodiments, the compound is selected from the group consisting of retinoic acid, phenylbutyrate, all-trans-retinoic acid and active form vitamin D.[0253] Pharmaceutical articles comprising an anti-MET antibody composition or anti-MET antibody or antigen-binding portion thereof of the invention and at least one other agent (e.g., a chemotherapeutic, anti-neoplastic, or anti-angiogenic agent) may be used as a combination treatment for simultaneous, separate or successive administration in cancer therapy. The other agent may by any agent suitable for treatment of the particular cancer in question, for example, an agent selected from the group consisting of alkylating agents, e.g., platinum derivatives such as cisplatin, carboplatin and/or oxaliplatin; plant alkoids, e.g., paclitaxel, docetaxel and/or irinotecan; antitumor antibiotics, e.g., doxorubicin (adriamycin), daunorubicin, epirubicin, idarubicin mitoxantrone, dactinomycin, bleomycin, actinomycin, luteomycin, and/or mitomycin; topoisomerase inhibitors such as topotecan; and/or antimetabolites, e.g., fluorouracil and/or other fluoropyrimidines.[0254] It is also contemplated that an anti-MET antibody or antigen-binding portion thereof or anti-MET antibody composition of the invention may be used in adjunctive therapy in connection with tyrosine kinase inhibitors. These are synthetic, mainly quinazoline-derived, low molecular weight molecules that interact with the intracellular tyrosine kinase domain of receptors and inhibiting ligand-induced receptor phosphorylation by competing for the intracellular Mg-ATP binding site. Pharmaceutical articles comprising an antibody composition of the invention and at least one TKI targeting MET thus may also be used as a combination treatment for simultaneous, separate or successive administration in cancer therapy.[0255] In certain aspects, the antibody compositions and antibodies and antigen-binding portions thereof of the invention may be administered in combination with another inhibitor of the MET pathway, which may target MET or HGF. In some embodiments, the inhibitor is selected from the group consisting of, but not limited to, AMG 102, AMG 208, AMG 458,ARQ 197, AV299, BAY-853474, CGEN241, DN30, E7050, EMD 1204831, EMD 1214063, INCB28060, JNJ38877605, K252a, LY-2875358, MGCD265, MK-2461, MP-470, NK4, OA- 5D5, PF-02341066, PF-04217903, PF-02341066, PHA-665752, SGX-523, SU5416, SU11274, TAK701, XL184, XL880, cabozantinib, crizotinib, ficlatuzumab, foretinib, golvatinib, onartuzumab, rilotumumab, and tivantinib.[0256] In some embodiments, the antibody compositions and antibodies and antigen- binding portions thereof of the invention may be administered in combination with an ErbB inhibitor (such as gefitinib or erlotinib) or a heat shock protein 90 (hsp90) inhibitor (such as 17-AAG).
PCT/IB2015/002110 WO 2016/042412 id="p-257" id="p-257" id="p-257" id="p-257" id="p-257"
id="p-257"
[0257]In other embodiments, the antibody compositions and antibodies and antigen- binding portions thereof of the invention may be used in combination with other antibody therapeutics, e.g., an antibody against VEGF (e.g., Avastin®). In yet other embodiments, the antibody compositions of the present invention may be used in combination with an agent known to stimulate cells of the immune system, such combination treatment leading to enhanced immune-mediated enhancement of the efficacy of the antibody compositions of the invention. Examples of such immune-stimulating agents include recombinant interleukins (e.g., IL-21 and IL-2). [0258]It is understood that the antibody compositions and antibodies and antigen-binding portions thereof of the invention may be used in a method of treatment as described above, may be for use in a treatment as described above, and/or may be for use in the manufacture of a medicament for a treatment as described above, Dose and Route of Administration id="p-259" id="p-259" id="p-259" id="p-259" id="p-259"
id="p-259"
[0259]The antibody compositions of the invention will be administered in an effective amount for treatment of the condition in question, i.e., at dosages and for periods of time necessary to achieve a desired result. A therapeutically effective amount may vary according to factors such as the particular condition being treated, the age, sex and weight of the patient, and whether the antibodies are being administered as a stand-alone treatment or in combination with one or more additional anti-cancer treatments. [0260]Dosage regimens may be adjusted to provide the optimum desired response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.Dosage unit form, as used herein, refers to physically discrete units suited as unitary dosages for the patients/subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are generally dictated by and directly dependent on (a) the unique characteristics of the chemotherapeutic agent and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals. [0261]Thus, the skilled artisan would appreciate, based upon the disclosure provided herein, that the dose and dosing regimen is adjusted in accordance with methods well- known in the therapeutic arts. That is, the maximum tolerable dose can be readily established, and the effective amount providing a detectable therapeutic benefit to a patient PCT/IB2015/002110 WO 2016/042412 may also be determined, as can the temporal requirements for administering each agent to provide a detectable therapeutic benefit to the patient. Accordingly, while certain dose and administration regimens are exemplified herein, these examples in no way limit the dose and administration regimen that may be provided to a patient in practicing the present invention. [0262]It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated, and may include single or multiple doses. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the embodied composition. Further, the dosage regimen with the compositions of this invention may be based on a variety of factors, including the type of disease, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the particular antibody employed. Thus, the dosage regimen can vary widely, but can be determined routinely using standard methods. For example, doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and/or laboratory values. Thus, the present invention encompasses intra-patient dose-escalation as determined by the skilled artisan. Determining appropriate dosages and regimens are well-known in the relevant art and would be understood to be encompassed by the skilled artisan once provided the teachings disclosed herein. [0263]It is contemplated that a suitable dose of an antibody composition of the invention will be in the range of 0.1-100 mg/kg, such as about 0.5-50 mg/kg, e.g., about 1-20 mg/kg. The antibody composition may for example be administered in a dosage of at least 0.mg/kg, e.g., at least 0.5 mg/kg, such as at least 1 mg/kg, e.g., at least 1.5 mg/kg, such as at least 2 mg/kg, e.g., at least 3 mg/kg, such as at least 4 mg/kg, e.g., at least 5 mg/kg; and e.g., up to at most 50 mg/kg, such as up to at the most 30 mg/kg, e.g., up to at the most mg/kg, such as up to at the most 15 mg/kg. Administration will normally be repeated at suitable intervals, e.g., once every week, once every two weeks, once every three weeks, or once every four weeks, and for as long as deemed appropriate by the responsible doctor, who may optionally increase or decrease the dosage as necessary. [0264]An effective amount for tumor therapy may be measured by its ability to stabilize disease progression and/or ameliorate symptoms in a patient, and preferably to reverse disease progression, e.g., by reducing tumor size. The ability of an antibody or composition of the invention to inhibit cancer may be evaluated by in vitro assays, e.g., as described in the examples, as well as in suitable animal models that are predictive of the efficacy in human tumors. Suitable dosage regimens will be selected in order to provide an optimum therapeutic response in each particular situation, for example, administered as a single bolus PCT/IB2015/002110 WO 2016/042412 or as a continuous infusion, and with possible adjustment of the dosage as indicated by the exigencies of each case.
Diagnostic Uses and Compositions id="p-265" id="p-265" id="p-265" id="p-265" id="p-265"
id="p-265"
[0265]The antibodies of the present invention also are useful in diagnostic processes (e.g., in vitro, ex vivo). For example, the antibodies can be used to detect and/or measure the level of MET in a sample from a patient (e.g., a tissue sample, or a body fluid sample such as an inflammatory exudate, blood, serum, bowel fluid, saliva, or urine). Suitable detection and measurement methods include immunological methods such as flow cytometry, enzyme-linked immunosorbent assays (ELISA), chemiluminescence assays, radioimmunoassay, and immunohistology. The invention further encompasses kits (e.g., diagnostic kits) comprising the antibodies described herein. [0266]In order that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner. [0267]All publications, patents, and patent applications cited in this specification are incorporated herein by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended embodiments.
Examples Example 1: Cloning of Anti-MET Antibodies id="p-268" id="p-268" id="p-268" id="p-268" id="p-268"
id="p-268"
[0268]Anti-MET antibodies were obtained using the Symplex™ procedure essentially as described in WO 2005/042774. Briefly, BALB/c, C57 and C3H mice were immunized bi- weekly with human cancer cell lines over-expressing MET (HCT-116), recombinant human MET protein (Sino Biologicals), recombinant human MET protein pre-incubated with ligand (HGF), or trypsin-digested MET. Murine plasma cells obtained from spleens and inguinal lymph nodes were FACS sorted, and linkage of VH and VL coding sequences was performed on the sorted plasma cells, facilitating cognate pairing of the sequences, utilizing a two-step PCR procedure based on a one-step multiplex overlap-extension RT-PCR followed by nested PCR. The principle for linkage of cognate VH and VL sequences is described in detail in WO 2005/042774 and in Meijer et al., J Mol Biol 358(3):764-72 (2006). [0269]In order to identify antibodies with binding specificity to MET, the VH and VL coding sequences obtained above were expressed as full-length antibodies. This involved insertion PCT/IB2015/002110 WO 2016/042412 of the repertoire of VH and VL coding pairs into an expression vector and transfection into a host cell using the method described in WO 2012/059858. [0270]The specificity of the produced antibodies was determined by ELISA using as antigen either the extracellular domain of the MET protein or the extracellular domain of the MET protein translationally fused to a human immunoglobulin Fc domain. Nunc MaxiSorp plates (Cat. No. 464718) were coated with 1 pg/ml of the recombinant MET protein diluted in PBS at 4°C overnight. The plates were washed once with PBS + 0.05% Tween 20 (PBS-T) prior to blocking in 50 pi 2% Milk- PBS-T. The plates were washed once again with PBS-T, then 20 pi of 2% milk-PBS-T. 10 pi of supernatants from the FreeStyle293 transfectants were added and incubated for 1 hour at room temperature, after which the plates were washed once with PBS-T. Secondary antibody (HRP-Goat-anti-human kappa light chain, Serotec, Cat. No. STAR 100P) diluted 1:25000 in 2% milk-PBS-T was added to detect the antibodies bound to the wells and incubated for 1 hour at room temperature. The plates were washed once in PBS-T before addition of 25 pi substrate (Kem-En-Tec Diagnostics, Cat. No. 4518) and incubation for 5 min. 25 pi 1M sulphuric acid was added after the incubation to stop the reaction. Specific signal was detected on an ELISA reader at 450 nm. From the ELISA data, positive antibody clones were identified and selected for sequence analysis and validation of binding to MET.
Example 2: Screening of Functional Anti-MET Antibody Mixtures id="p-271" id="p-271" id="p-271" id="p-271" id="p-271"
id="p-271"
[0271]This example describes in vitro testing of chimeric monoclonal antibodies targeting MET and mixtures of these monoclonal antibodies to identify lead candidates. The monoclonal antibodies and mixtures were evaluated for their ability to inhibit the growth of the cancer cell lines EBC1, MKN45, OE33 and SNU5.
Methods id="p-272" id="p-272" id="p-272" id="p-272" id="p-272"
id="p-272"
[0272]Mouse-derived antibodies targeting human MET were assayed for their ability to inhibit growth of human cancer cell lines in vitro. The monoclonal antibodies and 2-antibody mixtures (1:1 mixtures of two monoclonal antibodies) were diluted to a final total antibody concentration of 100 pg/ml in RPMI 1640 Glutamax media supplemented with 2% FBS and 1% P/S, yielding a final concentration of 5 pg/ml. Relevant numbers of cells (EBC1: 15cells/well, MKN45: 2000 cells/well, OE33 4300 cells/well and SNU5: 800 cells/well) were then added to the experimental wells in a 384 well plate, and incubated with antibodies for days in a humidified incubator at 37°C. WST-1 reagent was subsequently added to the plates, and incubated for one hour at 37°C. The absorbance was measured at 450 nm and 620 nm (reference wavelength) using an ELISA reader. The absorbance at 620 nM was PCT/IB2015/002110 WO 2016/042412 subtracted from the absorbance at 450 nM. The amount of metabolically active cells (MAC) was calculated as a percentage of the untreated control as follows: x !Of f fJSissp.■—■ ין It is assumed that the metabolic activity correlates with the number of viable cells, meaning that a lower %MAC corresponds to a higher level of cell growth inhibition by the antibodies.
Results id="p-273" id="p-273" id="p-273" id="p-273" id="p-273"
id="p-273"
[0273]Antibody mixtures were ranked based on their ability to inhibit cell growth among a panel of cancer cell lines. The viability results from monoclonal antibodies and mixtures of two antibodies on the metabolic activity of cell lines EBC1, MKN45, OE33 and SNU5 are shown in Table 3. Seven different mixtures of two antibodies inhibit metabolic activity to an average below 50%. [0274]Among the most efficacious mixtures, the combination of the two antibodies 90and 9338 exhibited a broad cell growth inhibitory activity. Interestingly, each antibody alone exhibited lower efficacy than the combination, suggesting that the two antibodies can act synergistically. In addition to 9006+9338, it may be seen from Table 3 that other highly efficacious mixtures include 9206+9232, 8955+9338, 9206+9338, 9006+9232, 8955+9006, 8955+9096 and 8955+9232. Further, it will be apparent that these top eight mixtures are based on relatively few individual monoclonal antibodies, in particular 8955, 9006, 9232, 9338 and 9206.
Table 3. Anti-proliferative effect of monoclonal antibodies and antibody mixtures Metabolic activty of cells treated with mAb or mAb mixture(% of untreated control)mAb or mAb mixtureSequenceCluster(s)SNU5 OE33 MKN45 EBC1 Average Rank9206 + 9232 008 + 007 32 70 32 37 43 19006 + 9338 018 + 004 33 54 31 54 43 28955 + 9338 029 + 004 33 60 24 63 45 39206 + 9338 008 + 004 39 52 34 55 45 49006 + 9232 018 + 007 36 72 24 59 48 58955 + 9006 029 + 018 46 62 25 61 48 68955 + 9096 029 + 018 55 41 40 59 49 78955 + 9232 029 + 007 46 66 25 69 51 89206 + 9217 008 + 012 35 60 54 99 62 98955 + 9206 029 + 008 39 71 57 82 62 109044 + 9111 011 + 009 47 77 37 91 63 119111 + 9217 009 + 012 40 101 24 93 64 129111 + 9232 009 + 007 67 83 53 63 66 13 PCT/IB2015/002110 WO 2016/042412 Metabolic activty of cells treated with mAb or mAb mixture(% of untreated control)mAb or mAb mixtureSequenceCluster(s)SNU5 OE33 MKN45 EBC1 Average Rank9006 + 9111 018 + 009 31 101 36 98 66 149111 + 9206 009 + 008 39 79 53 96 67 159006 + 9154 018 + 009 34 91 49 98 68 169096 + 9232 018 + 007 50 79 54 94 69 179184 + 9217 009 + 012 45 103 30 101 70 189184 + 9206 009 + 008 30 88 63 97 70 199173 + 9232 028 + 007 51 90 84 57 71 209006 + 9184 018 + 009 35 105 44 107 73 219154 + 9217 009 + 012 52 98 36 105 73 229212 + 9232 025 + 007 57 92 90 64 76 239154 + 9206 009 + 008 48 66 87 103 76 249096 + 9184 018 + 009 47 80 71 108 76 259096 + 9111 018 + 009 43 107 65 94 77 269173 + 9340 028 + 072 40 98 79 98 79 279044 + 9184 011 + 009 57 104 54 102 79 289184 + 9232 009 + 007 92 100 62 63 79 299111 + 9173 009 + 028 47 103 69 103 80 309111 + 9133 009 + 028 54 103 64 106 82 319006 + 9122 018 + 031 46 112 50 118 82 329096 + 9338 018 + 004 50 57 100 120 82 339173 028 38 99 83 108 82 349006 + 9146 018 + 036 61 75 79 118 83 359146 + 9173 036 + 028 48 115 66 105 83 369173 + 9184 028 + 009 44 113 70 108 84 378820 + 9006 044 + 018 55 103 65 115 85 389044 + 9154 011 + 009 59 100 67 113 85 398908 + 9006 032 + 018 42 110 50 138 85 409173 + 9206 028 + 008 47 107 74 112 85 419173 + 9212 028 + 025 46 99 93 102 85 429133 + 9232 028 + 007 77 110 88 66 85 439044 + 9206 011 + 008 89 65 83 107 86 449146 + 9232 036 + 007 105 90 86 65 86 458955 + 9111 029 + 009 73 96 61 117 87 469006 + 9173 018 + 028 43 109 84 113 87 479154 + 9173 009 + 028 55 104 79 112 88 489154 + 9232 009 + 007 94 115 73 70 88 499006 + 9212 018 + 025 61 79 94 120 88 509096 + 9154 018 + 009 73 82 83 115 88 519006 018 68 73 90 122 88 529122 + 9206 031 + 008 67 110 56 125 89 53 PCT/IB2015/002110 WO 2016/042412 Metabolic activty of cells treated with mAb or mAb mixture(% of untreated control)mAb or mAb mixtureSequenceCluster(s)SNU5 OE33 MKN45 EBC1 Average Rank8820 + 8955 044 + 029 87 87 80 106 90 548908 + 9217 032 + 012 74 96 65 125 90 559006 + 9096 018 + 018 68 71 90 131 90 569133 + 9212 028 + 025 59 96 102 104 90 579122 + 9232 031 + 007 117 86 95 62 90 589006 + 9133 018 + 028 58 111 81 113 90 598955 + 9007 029 + 046 83 81 98 100 91 608908 + 8955 032 + 029 79 70 87 128 91 619096 + 9173 018 + 028 39 119 88 117 91 629133 + 9173 028 + 028 45 102 116 102 91 639217 + 9232 012 + 007 114 100 82 68 91 648955 + 9146 029 + 036 83 72 92 118 91 659122 + 9217 031 + 012 65 126 46 128 91 668955 + 9154 029 + 009 80 80 85 122 92 679133 + 9184 028 + 009 58 112 85 111 92 689006 + 9044 018 + 011 66 75 91 135 92 698955 + 8958 029 + 029 72 94 81 121 92 708820 + 9111 044 + 009 61 135 53 120 92 718955 + 9122 029 + 031 69 91 81 129 93 729096 + 9133 018 + 028 57 99 95 120 93 739006 + 9217 018 + 012 67 93 92 120 93 749111 + 9122 009 + 031 98 106 50 118 93 759146 + 9184 036 + 009 89 93 81 110 93 769212 + 9340 025 + 072 69 117 86 99 93 778820 + 9232 044 + 007 88 103 75 108 93 789146 + 9206 036 + 008 79 75 101 119 94 799044 + 9122 011 + 031 80 109 63 124 94 809006 + 9340 018 + 072 70 78 101 127 94 819173 + 9338 028 + 004 61 102 92 120 94 828820 + 9184 044 + 009 67 121 60 128 94 839006 + 9206 018 + 008 81 78 101 117 94 849173 + 9217 028 + 012 58 124 72 123 94 859133 + 9206 028 + 008 67 102 95 112 94 869006 + 9007 018 + 046 61 92 81 142 94 879212 025 86 90 101 100 94 889232 + 9338 007 + 004 86 93 77 122 94 899133 028 68 102 99 109 95 908820 + 9206 044 + 008 58 129 69 123 95 918955 + 9340 029 + 072 81 70 97 133 95 929111 + 9146 009 + 036 93 108 74 109 96 93 PCT/IB2015/002110 WO 2016/042412 Metabolic activty of cells treated with mAb or mAb mixture(% of untreated control)mAb or mAb mixtureSequenceCluster(s)SNU5 OE33 MKN45 EBC1 Average Rank9096 + 9206 018 + 008 70 79 98 138 96 949133 + 9146 028 + 036 67 100 112 106 96 959096 + 9122 018 + 031 49 121 77 139 96 968906 + 9232 056 + 007 89 111 74 112 97 979133 + 9340 028 + 072 69 123 93 101 97 988908 + 9232 032 + 007 95 100 76 116 97 998955 + 9184 029 + 009 74 96 87 130 97 1009044 + 9232 011 + 007 85 112 82 110 97 1018955 + 9212 029 + 025 105 75 91 118 97 1028899 + 8955 029 + 029 78 77 97 137 97 1039111 + 9212 009 + 025 89 91 101 109 97 1048820 + 9173 044 + 028 46 158 77 112 98 1059096 + 9146 018 + 036 81 78 101 133 98 1069096 + 9212 018 + 025 83 86 98 127 98 1078955 + 9133 029 + 028 85 96 89 126 99 1089212 + 9338 025 + 004 66 118 93 119 99 1098820 + 9338 044 + 004 90 96 91 119 99 1108906 + 9217 056 + 012 82 106 81 128 99 1119206 008 90 86 97 125 99 1128955 + 9173 029 + 028 69 113 94 123 100 1139007 + 9232 046 + 007 109 99 77 114 100 1148908 + 9111 032 + 009 93 114 79 114 100 1159111 + 9184 009 + 009 88 99 100 115 100 1169007 + 9217 046 + 012 89 97 90 126 100 1178955 029 100 68 101 132 100 1189154 + 9184 009 + 009 90 102 91 119 101 1199122 + 9173 031 + 028 75 131 80 116 101 1208820 + 9096 044 + 018 82 95 86 140 101 1219111 + 9154 009 + 009 93 103 91 117 101 1229096 + 9340 018 + 072 80 81 105 139 101 1238908 + 9206 032 + 008 90 108 80 127 101 1249206 + 9212 008 + 025 91 77 114 124 101 1258820 + 9122 044 + 031 63 158 61 125 102 1269146 036 119 92 100 100 103 1279206 + 9340 008 + 072 103 94 94 120 103 1288906 + 9006 056 + 018 66 142 52 152 103 1298820 + 9133 044 + 028 63 152 80 116 103 1308902 + 8955 029 + 029 72 87 105 149 103 1319232 + 9340 007 + 072 115 93 85 120 103 1328955 + 9026 029 + 029 91 75 101 146 104 133 PCT/IB2015/002110 WO 2016/042412 Metabolic activty of cells treated with mAb or mAb mixture(% of untreated control)mAb or mAb mixtureSequenceCluster(s)SNU5 OE33 MKN45 EBC1 Average Rank9146 + 9212 036 + 025 110 93 106 106 104 1349111 009 97 96 102 119 104 1358955 + 9217 029 + 012 94 97 91 134 104 1368820 + 9154 044 + 009 72 148 73 123 104 1378906 + 9206 056 + 008 90 126 75 127 104 1388820 + 9217 044 + 012 91 151 65 112 105 1399044 + 9217 011 + 012 103 102 93 120 105 1409232 007 115 100 84 121 105 1419340 072 113 99 95 114 105 1429217 + 9338 012 + 004 83 124 89 126 106 1439096 018 90 86 103 144 106 1449184 + 9212 009 + 025 98 103 108 114 106 1459007 + 9173 046 + 028 67 153 95 109 106 1468908 + 9184 032 + 009 85 127 90 123 106 1479111 + 9340 009 + 072 107 109 101 111 107 1489044 + 9133 011 + 028 91 132 87 119 107 1499133 + 9154 028 + 009 90 123 101 117 108 1508820 + 9044 044 + Oil 97 138 79 117 108 1518820 + 9212 044 + 025 82 143 87 119 108 1529122 + 9184 031 + 009 96 140 73 124 108 1539007 + 9206 046 + 008 89 113 95 136 108 1549184 009 101 110 101 123 109 1559338 004 100 98 96 141 109 1568906 + 9111 056 + 009 112 120 84 122 110 1579111 + 9338 009 + 004 107 121 90 121 110 1589146 + 9340 036 + 072 112 137 90 100 110 1599133 + 9338 028 + 004 89 133 96 120 110 1608820 + 8906 044 + 056 86 148 80 126 110 1619217 012 96 108 100 135 110 1629044 + 9338 Oil + 004 101 101 103 135 110 1638908 + 9154 032 + 009 98 132 87 124 110 1649007 + 9096 046 + 018 88 96 96 160 110 1659212 + 9217 025 + 012 80 121 111 129 110 1669044 + 9173 011 + 028 72 150 95 126 111 1679007 + 9154 046 + 009 100 135 83 126 111 1689154 + 9340 009 + 072 113 117 85 131 111 1699338 + 9340 004 + 072 103 106 103 134 112 1709096 + 9217 018 + 012 106 108 96 137 112 1719007 + 9184 046 + 009 85 137 95 131 112 1729007 + 9340 046 + 072 109 112 95 133 112 173 PCT/IB2015/002110 WO 2016/042412 Metabolic activty of cells treated with mAb or mAb mixture(% of untreated control)mAb or mAb mixtureSequenceCluster(s)SNU5 OE33 MKN45 EBC1 Average Rank9044 + 9340 011 + 072 92 120 100 138 113 1749184 + 9340 009 + 072 109 132 99 110 113 1759122 + 9133 031 + 028 92 133 105 124 113 1768955 + 9044 029 + 011 102 100 102 151 114 1778906 + 9044 056 + 011 96 122 86 151 114 1789007 + 9122 046 + 031 108 135 85 127 114 1798820 + 9146 044 + 036 101 153 84 119 114 1808908 + 9122 032 + 031 131 132 76 117 114 1818906 + 9133 056 + 028 92 155 92 118 114 1828908 + 9133 032 + 028 95 158 96 110 115 1838908 + 9096 032 + 018 85 117 88 169 115 1849217 + 9340 012 + 072 106 137 87 129 115 1859133 + 9217 028 + 012 99 135 98 127 115 1868906 + 9173 056 + 028 72 185 79 122 115 1879007 + 9146 046 + 036 108 146 79 127 115 1888906 + 9184 056 + 009 105 147 81 128 115 1899007 + 9111 046 + 009 108 138 86 129 115 1908908 + 9173 032 + 028 85 170 90 118 116 1918908 + 9044 032 + 011 87 135 81 159 116 1929044 + 9146 011 + 036 119 129 93 122 116 1938908 + 9212 032 + 025 120 125 95 123 116 1949007 + 9212 046 + 025 115 127 93 128 116 1959044 + 9212 011 + 025 133 117 96 118 116 1968820 + 9007 044 + 046 96 152 84 134 117 1979044 Oil 115 118 98 137 117 1988906 + 9122 056 + 031 122 148 78 120 117 1998908 + 9340 032 + 072 106 128 94 141 117 2009184 + 9338 009 + 004 124 129 92 126 118 2018906 + 9154 056 + 009 119 144 79 128 118 2029007 + 9044 046 + Oil 112 129 87 145 118 2038906 + 9212 056 + 025 133 136 86 120 119 2049146 + 9338 036 + 004 135 126 91 123 119 2058908 + 9338 032 + 004 106 129 101 139 119 2069007 + 9338 046 + 004 96 136 101 144 119 2079154 009 123 119 97 138 119 2089122 + 9338 031 + 004 129 123 99 126 119 2098820 + 8908 044 + 032 110 154 83 131 119 2108820 044 111 157 88 121 119 2119007 + 9133 046 + 028 92 166 101 119 120 2129146 + 9217 036 + 012 123 128 104 126 120 213 PCT/IB2015/002110 WO 2016/042412 Metabolic activty of cells treated with mAb or mAb mixture(% of untreated control)mAb or mAb mixtureSequenceCluster(s)SNU5 OE33 MKN45 EBC1 Average Rank9122 + 9154 031 + 009 112 151 88 129 120 2148906 + 9096 056 + 018 93 127 86 176 120 2158906 + 9146 056 + 036 102 176 83 121 121 2169154 + 9212 009 + 025 135 111 110 128 121 2178908 + 9146 032 + 036 126 150 90 121 122 2188906 + 9340 056 + 072 106 157 95 129 122 2199146 + 9154 036 + 009 116 130 111 132 122 2209154 + 9338 009 + 004 137 128 88 139 123 2218820 + 9340 044 + 072 113 143 96 140 123 2229007 046 122 141 94 139 124 2238906 + 8908 056 + 032 93 147 98 157 124 2249122 + 9212 031 + 025 130 138 110 123 125 2258908 032 135 134 96 140 126 2268906 + 9338 056 + 004 119 166 93 129 127 2279122 + 9146 031 + 036 146 128 109 128 128 2288906 056 127 153 95 136 128 2298906 + 9007 056 + 046 84 157 100 172 128 2309044 + 9096 011 + 018 124 138 98 155 129 2319122 031 142 134 106 136 130 2329122 + 9340 031 + 072 146 150 98 125 130 2338908 + 9007 032 + 046 109 152 101 169 133 2348906 + 8955 056 + 029 136 167 99 150 138 235 Example 3: Humanization of the 9006 and 9338 antibodies id="p-275" id="p-275" id="p-275" id="p-275" id="p-275"
id="p-275"
[0275]This example describes humanization of the murine antibody framework regions of the 9006 and 9338 antibodies. Antibody humanization is performed to produce a molecule with minimal immunogenicity when applied to humans, while retaining the specificity and affinity of the parental non-human antibody.
Methods id="p-276" id="p-276" id="p-276" id="p-276" id="p-276"
id="p-276"
[0276]Humanization of the 9006 and 9338 antibodies was performed using the "CDR grafting" approach. First, the original murine germline genes were identified by blasting the V gene sequences of 9006 (Figure 26) and 9338 (Figure 27) against mouse germline V and J gene databases. This indicated that the closest mouse germline genes were IGHV9- 1*02/IGHJ4*01 and IGKV8-28*01/IGKJ2*01 for the variable heavy and variable light genes of 9006, respectively. Similarly, the closest mouse germline genes were IGHV1- 4*01/IGHJ3*01 and IGKV4-79*01/IGKJ4*01 for the variable heavy and variable light genes PCT/IB2015/002110 WO 2016/042412 of 9338, respectively. Second, the antibody VH and VL genes were aligned against the murine germlines to identify somatic mutations in the framework regions that may play a role in antibody function and/or structure. Such residues may be included in the final humanized antibody genes as so-called "back mutation" residues. The 9006 and 9338 variable antibody sequences were then blasted against human immunoglobulin databases to identify the closest human germlines whose framework regions will be used for the antibody humanization. For the antibody 9006, the retained human germlines were IGHV7-4- 1*02/IGHJ6*01 and IGKV4-1*01/IGKJ2*01 for the variable heavy and variable light genes, respectively. For the antibody 9338, the retained human germlines were IGHV1- 69*08/IGHJ1*01 and IGKV3-11*01/IGKJ2*01 for the variable heavy and variable light genes, respectively. Finally, for each antibody, the CDR regions from the chimeric antibodies were grafted onto the selected human framework and J gene segments. The CDR sequences were assigned in accordance with IMGT® definitions.
Results id="p-277" id="p-277" id="p-277" id="p-277" id="p-277"
id="p-277"
[0277]The final humanized 9006 and 9338 antibody sequences are shown in Figure and Figure 29, respectively.
Example 4: Cloning of anti-MET reference antibody analogs id="p-278" id="p-278" id="p-278" id="p-278" id="p-278"
id="p-278"
[0278]This example lists the sources of the amino acid sequences and the final antibody format used for generation of anti-MET reference antibody analogs. Some of the listed antibodies have been extensively characterized and have well-defined epitopes. A number of the antibodies have also entered clinical evaluation.
Methods id="p-279" id="p-279" id="p-279" id="p-279" id="p-279"
id="p-279"
[0279]The amino acid sequences encoding the variable heavy and light chain domains of the antibody analogs in Table 4 were obtained from the listed patents or patent applications. The protein sequences were reverse translated to DNA sequences with human codon usage. The corresponding DNA sequences were gene synthesized and cloned into expression vectors containing constant human heavy or light chain domains, resulting in expression of full-length antibodies. One exception was for the 5D5 antibody that was expressed as a Fab fragment. The human antibody isotype selected for expression is listed in the antibody format column together with additional mutations introduced in the Fc region where applicable. CHO cells were transfected with the corresponding expression plasmids using a standard protein expression system, with the exception of hybridoma clone HB- 12093, which was grown using standard hybridoma culturing technique. The corresponding antibody supernatants were purified using standard protein A purification column chromatography.
PCT/IB2015/002110 WO 2016/042412 Table 4. Listing of gene-synthesized antibody analogs and the corresponding antibody format. Antibody clone Research code Antibody format Reference 224G11 ABT-700, h224G11 Recombinant lgG1 EP2014681A1223C4 N.A. Recombinant lgG1 EP2014681A1 C8-H241LY2875358, LA480, emibetuzumabRecombinant lgG4 (S228P, F234A, L235A)WO2010059654A1 36C4 ARGX-111 Recombinant lgG1 US2012/0148607A1 5D5OA-5D5, MetMAb, onartuzumabRecombinant lgG1 Fab US 7476724 B2 13-MET N.A. Recombinant lgG1 W02009/142738 A228-MET N.A. Recombinant lgG1 W02009/142738 A2HB-12093 N.A. Mouse Hybridoma EP 0922102 Example 5: Epitope binning of MET antibodies id="p-280" id="p-280" id="p-280" id="p-280" id="p-280"
id="p-280"
[0280]This example illustrates how the MET antibodies were grouped into epitope bins based on pairwise competition patterns. Antibodies belonging to different epitope bins recognize different epitopes on the MET extracellular domain (ECD).
Methods id="p-281" id="p-281" id="p-281" id="p-281" id="p-281"
id="p-281"
[0281]Investigation of pairwise antibody competition was performed by Bio-layer Interferometry (BLI) analysis using an Octet QK384 instrument (Fortebio, USA). Commercially available human MET Fc fusion protein (R&D Systems) was captured on anti- human Fc sensor chips (Fortebio, USA) and residual anti-Fc sites blocked with Herceptin negative control antibody. The antigen coated surface was saturated with an anti-MET antibody concentration of 80 pg/ml followed by evaluation of pairwise anti-MET antibody combinations in competitive binding experiments. The sensor surface was regenerated by incubation with 10 mM glycine-HCI, pH 1.5 and reused for a new competition cycle.
Results id="p-282" id="p-282" id="p-282" id="p-282" id="p-282"
id="p-282"
[0282]The competition pattern of the 13 tested MET antibodies is presented in Figure 1. The MET antibodies were found to group in 12 distinct epitope bins. The antibody Hu90was found to bind a distinct epitope that overlapped with C8-H241. However the epitope was different compared to C8-H241, since C8-H241was also blocked by Hu9338 and 36C4, while Hu9006 was not. Consequently, Hu9006 and C8-H241 were assigned to different epitope bins. The epitope of Hu9338 overlapped with 36C4 and both antibodies showed PCT/IB2015/002110 WO 2016/042412 identical competition patterns with other antibodies in the tested panel, and these were consequently assigned to the same epitope bin. [0283]Antibodies 224G11,28-MET, 5D5, 9206 & 13-MET showed in some instances unidirectional inhibition. This observed phenomenon could be caused by allosteric effects and was observed in repeated competition experiments.
Example 6: Analysis of MET Antibodies for HGF ligand blocking activity id="p-284" id="p-284" id="p-284" id="p-284" id="p-284"
id="p-284"
[0284]This example illustrates how the panel of anti-MET antibodies was analyzed for HGF ligand blocking activity by performing a competition assay using Bio-Layer Interferometry analysis.
Methods id="p-285" id="p-285" id="p-285" id="p-285" id="p-285"
id="p-285"
[0285]Investigation of HGF ligand blocking activity was performed by Bio-Layer Interferometry (BLI) analysis using an Octet QK384 instrument (Fortebio, USA). Commercially available human MET Fc fusion protein (R&D Systems) was captured on anti- human Fc sensor chips (Fortebio, USA) and residual anti-Fc sites blocked with Herceptin negative control antibody. Next the antigen coated surface was saturated with an anti-MET antibody concentration of 80 pg/ml (533 nM), except for 5D5 Fab fragment, which was diluted to 26.7 pg/ml (533 nM). After MET saturation with antibody HGF ligand blocking activity was assessed by incubation with human HGF ligand (R&D Systems) tested at pg/ml (222 nM). Herceptin lgG1 was used as a negative control antibody.
Results id="p-286" id="p-286" id="p-286" id="p-286" id="p-286"
id="p-286"
[0286]The result of the competition analysis is presented in Table 5 below. Antibody Hu9006 and Hu9338 were both found to inhibit HGF ligand binding by approx. 80%, while 5D5 Fab was found to fully block HGF binding (100%). When equimolar concentrations of Hu9006 and Hu9338 were mixed (80 pg/ml total concentration, 533 nM), HGF ligand binding was inhibited by approx. 90%. Consequently, more efficient HGF ligand blocking activity was obtained by mixing antibodies Hu9006 and Hu9338 1:1.The antibodies C8-H241 and 36Cwere found to inhibit HGF ligand binding by approx. 80 and 75%, respectively, while antibodies 13-MET and 28-MET blocked HGF binding by approx. 80 and 50%, respectively. The agonistic antibody 5882 and the negative control antibody Herceptin did not block HGF binding (3-7% HGF binding inhibition).
Table 5. HGF binding inhibition after MET antibody saturation.
Antibody % HGF binding inhibition Hu9006 78Hu9338 81 PCT/IB2015/002110 WO 2016/042412 Antibody % HGF binding inhibition 5882 3Hu9006+ Hu9338 89C8-H241 8136C4 74224G11 20223C4 6913-MET 8128-MET 50HB-12093 95D5 Fab 10012398 849206 62Herceptin 6 Example 7: Epitope mapping ofAnti-MET Antibodies id="p-287" id="p-287" id="p-287" id="p-287" id="p-287"
id="p-287"
[0287]This example illustrates how the binding epitopes of the MET antibodies of the invention were mapped to blade 2 or 3 in the SEMA-a domain, by analyzing binding to chimeric MET constructs expressed on cells. The example also illustrates how the epitopes of the antibodies of the invention are distinct compared to the tested reference antibody analogs.
Methods id="p-288" id="p-288" id="p-288" id="p-288" id="p-288"
id="p-288"
[0288]The human MET receptor consists of an extracellular domain of 907 amino acids (residues 25-932). The extracellular domain can be subdivided into the SEMA domain (residues 27-515), a cysteine rich Plexin Semaphorin Integrin domain (PSI domain, residues 520-561) and four immunoglobulin like domains defined by the following amino acid sequences. IPT1: AA 563-655. IPT2: AA 657-739. IPT3: AA 742-836. IPT4: AA 837-932. The domain definitions are described in Gherardi et al., Proc Natl Acad Sci USA.100(21 ):12039-44 (2003) and Uniprot entry P08581. The SEMA domain consists of seven beta sheets (blades) that fold into of a seven-bladed propeller structure (Stamos J. et al., EMBO J. 23:2325-2335. (2004)). A furin cleavage site is present at position 307-308, dividing the SEMA domain into a and 3 chains. The SEMA-a domain is encoded by amino acid residues 27-307 composing blades 1-4 and the SEMA-3 domain is encoded by amino acid residues 308-515 composing blades 5-7. The SEMA-a domain contains a binding site for the 3־chain of the HGF ligand while the MET binding site of the HGF a-chain remains PCT/IB2015/002110 WO 2016/042412 elusive (Merchant et al., Proc Natl Acad Sci USA. 110(32):E2987-96 (2013)). A single report claims that the IPT3 and IPT4 domains of MET ECD also mediate high affinity HGF binding (Basilico et al., J Biol Chem. 283(30):21267-21277 (2008)). [0289]The mRNA sequence of human MET isoform 1 was downloaded from NCBI (ACCESSION NM_000245.2; the amino acid sequence is represented in SEQ ID NO: 1). Human MET also exists in a different isoform (isoform 2) where 19 amino acids (STWWKEPLNIVSFLFCFAS (SEQ ID NO: 2)) replace S755 in the IPT domain 3. The amino acid sequence of isoform 2 is listed as SEQ ID NO: 2. The full-length chicken and murine MET protein sequences including leader peptide sequences were downloaded from NCBI (ACCESSION NP_990543 (SEQ ID NO: 3) and NP_032617 (SEQ ID NO: 4) respectively). Chimeric human/chicken domain exchange variants of the extracellular domain (ECD), where each domain or subdomain was sequentially replaced with chicken DNA sequence, were gene synthesized together with fully human, murine or chicken MET ECD genes. Chimeric constructs where each of the seven blades in the SEMA domain were sequentially exchanged from human to mouse sequence were also synthesized. [0290]The constructs used for determining the blade binding specificity were as follows (numbers refer to sequence exchanged to mouse sequence): Mouse blade 1: AA 25-83. Mouse blade 1-2: AA 25-162. Mouse blade 1-3: AA 25-233. Mouse blade 1-4: AA 25-295. Mouse blade 1-5: AA 25-430. Mouse blade 1-6: AA 25-479. Mouse blade 1-7: AA 25-513. The reverse constructs were also made. Mouse PSI-IPT4: (AA 515-932). Mouse blade 7- IPT4: (AA 480-932). Mouse blade 6-IPT4: (AA 431-932). Mouse blade 5b-IPT4: (AA 382- 932). Mouse blade 5a-IPT4: (AA 293-932). Mouse blade 4-IPT4: (AA 234-932). Mouse blade 3-IPT4: (AA 163-932). Mouse blade 2-IPT4: (AA 84-932). Blades 1-4 are located in the SEMA -a subdomain and blades 5-7 in the SEMA-3 subdomain. [0291]Recently other chimeric constructs where llama sequences were exchanged with human sequences in the MET SEMA domain have been described (Basilico C. et al. J. Clin Invest. 124:3172-3186 (2014)). These constructs were synthesized as well, but with the modification that the mouse sequence was inserted instead of the llama sequence, since the llama MET sequence was not publicly available. The sequence definitions for the additional chimeric proteins were as follows (AA numbers refer to sequence exchanged to mouse sequence): LS1: AA25-122, LS2: AA25-224, LS3: AA25-312, LS4: AA25-371, LS5: AA25- 473. LS1-3 reside in the SEMA-a subdomain and LS4-6 in the SEMA-3 subdomain. Finally, constructs where 15 AA of the human MET ECD sequence in the SEMA-a subdomain were sequentially exchanged to mouse sequence were synthesized for more detailed mapping of linear epitopes. For construct 109-120 only 11 amino acids were exchanged to mouse sequence. Each construct was designed to overlap with 2 amino acids, and in total 22 PCT/IB2015/002110 WO 2016/042412 constructs with up to 15 AA substitutions were made covering the human MET SEMA-a subdomain sequence after blade 1 (AA 89-313). [0292]All the synthesized chimeric or wild type constructs described above were subcloned into expression vectors containing a SV5 peptide tag, a glycine serine linker and the coding sequence for a glycosylphospha-tidylinositol (GPI) anchor resulting in C-terminal fusion of this cassette to the gene of interest (Bouquin T. et al., J. Biotechnol. 125:516-5(2006). The generated expression constructs were used for transient Freestyle™ transfection of HEK293 cells and the produced fusion proteins were targeted to the cell membrane via the GPI anchor. MET antibodies were analyzed for binding to transfected cells by flow cytometry using an iQue® Screener (IntelliCyt corporation). Antibodies were tested in an 8-point titration experiment using 3 fold dilutions beginning from 50 pg/ml and detection with an anti-human IgG (H+L) Alexa Fluor® 647 dye. The expression levels of the MET constructs were monitored by biotinylated anti-SV5 mAb MCA1360B and detection with Streptavidin APC Cy7. Cut-off values defined as the average fluorescence signal of all antibodies tested at 50 pg/ml to the negative control chicken or mouse MET construct + four standard deviations were employed to discriminate background binding from specific binding for the domain exchange or blade exchange constructs. Antibody binding to constructs where single amino acids or 15 amino acids were exchanged to mouse sequence were normalized to 5D5 binding tested at 3 pg/ml, since the mutations were located in the SEMA- a subdomain and shown not to influence the binding of 5D5 directed against the SEMA-subdomain.
Results id="p-293" id="p-293" id="p-293" id="p-293" id="p-293"
id="p-293"
[0293]The surface expression level of the wild type and chimeric human, chicken or mouse MET ECD constructs were evaluated with SV5 staining. All evaluated constructs expressed well and could be stained with the SV5 antibody, except the construct containing chicken SEMA-3 subdomain. The titers of each MET antibody binding to the constructs were evaluated (data not shown). A summary of the antibody binding to the different tested chimeric constructs is presented in Figure 2. A summary of the differential antibody binding to human MET ECD constructs where 15 AA segments in SEMA-a subdomain were sequentially exchanged to mouse is presented in Table 6, and a summary of differential antibody binding to human MET ECD constructs where surface exposed residues in SEMA- a subdomain were mutated to mouse sequence is presented in Table 7. Finally, a summary of all the epitope findings is shown in Table 8. [0294]All tested antibodies except 5D5 and 224G11 were found to bind the SEMA-a subdomain.
PCT/IB2015/002110 WO 2016/042412 id="p-295" id="p-295" id="p-295" id="p-295" id="p-295"
id="p-295"
[0295]Fine epitope mapping using the chimeric constructs introducing mutations in the SEMA -a domain illustrated that Hu9338 bound to a linear epitope located in blade 2 as illustrated by a significant loss of binding (36% binding compared to 5D5), when the sequence segment AA 99-113 was exchanged to mouse (Table 6). The epitope for Hu93was distinct and not found for the other antibodies in the tested MET panel. Hu9006 was found to bind to an epitope present in a fragment of blade 3 (AA 163-224). None of single amino acid point mutated MET constructs or MET constructs with 15 AA inserted mouse MET sequence showed significantly different binding of hu9006 compared to fully human MET ECD. Consequently, the epitope of hu9006 was distinct compared to the other members of the anti-MET antibody panel. The finding that Hu9338 and Hu9006 bound to epitopes located in blade 2 and 3 respectively was consistent with these antibodies being non-competitive and belonging to different epitope bins. [0296]The agonistic antibody 5882 was also found to bind to blade 3 (AA 163-224), but with contact residues at positions F206, D208, H209 & P210 as revealed by at least 50% or less binding compared to 5D5, when exchanging these positions to mouse sequence. Importantly, these closely located mutations did not significantly affect the binding of the other antibodies in the panel, illustrating that the strong agonistic activity of 5882 is related to binding the region defined by these 4 substitutions. [0297]The C8-H241 antibody was found to bind to epitopes located in both blade 2 and 3. While blade exchange constructs showed that this antibody bound an important epitope in blade 3 (AA 163-224), further epitope refinement could be obtained by the observed reduction of binding to constructs where 15 AA in blade 2 (AA 119-133) or 24 AA blade (AA 209-233) were exchanged to mouse sequence (68-30% binding respectively compared to 5D5). Finally, a contact residue identified in blade 3 (K223) resulting in only 19% binding compared to 5D5 indicated that the core epitope of the C8-H241 antibody is located in blade 3. The results were in good agreement with previously published data (Liu L. et al., Clin. Cancer. Res. 20:6059-6070 (2014)) showing that the linear epitopes of C8-H241 as determined by HD Exchange Mass Spectroscopy were present at positions 123-128, 144- 156, 192-195 and 220-227. [0298]Finally, we were able to map the epitope of 36C4 in finer detail. While Basilico et al. (Basilico C. et al. J. Clin Invest. 124:3172-3186 (2014)) described the epitope of 36C4 to be present in blade 2 & 3 (AA 98-199), we showed that the specificity can be divided into a linear epitope at position 129-143 in blade 2 (58% binding compared to 5D5) and a contact residue at position H209 in blade 3 (43% binding compared to 5D5). The contact residue at position H209 was also shared with the agonistic 5882 antibody, but since 5882 also bound three other closely located contact residues the binding and thus agonistic properties were clearly different.
PCT/IB2015/002110 WO 2016/042412 id="p-299" id="p-299" id="p-299" id="p-299" id="p-299"
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[0299]The crystal structure of 5D5 binding to the SEMA domain has previously been published (Merchant M. et al., Proc. Natl. Acad. Sci. USA. 110:E2987-E2996 (2013)), and the study showed that 5D5 recognized mainly blade 5 and 6 in the SEMA 3 sub domain. Key amino acid residues at positions Q328, R331, L337 and N338 were present in blade 5, and when mutated to mouse residues these significantly reduced binding affinity. This result is in agreement with our binding analysis that clearly showed that 5D5 recognized a crucial epitope in blade 5 (AA 313-371). [0300]We also found that the antibody 224G11 recognized the ITP1 domain in agreement with the information provided by Basilico and colleagues (Basilico C. et al. J. Clin Invest. 124:3172-3186 (2014).
Table 6. Antibody binding to human MET ECD constructs expressed on HEK293 cells, where 15 AA segments in the SEMA-a domain were sequentially exchanged to mouse. Construct Hu9338 Hu9006 C8-H241 36C4 5D5 Cetuximab MET 99-113 36 112 113 94 100 2MET 119-133 131 88 68 107 100 2MET 129-143 124 97 77 5 8 100 2MET 209-223 133 114 57 96 100 2MET 219-233 130 141 30 109 100 2human MET 144 137 135 162 100 1 Antibody binding is expressed as the percentage of 5D5 binding. Grey cells indicate less than 70% antibody binding compared to 5D5.
Table 7. Antibody binding to human MET ECD constructs expressed on HEK293 cells, where surface exposed residues in the SEMA-a domain were exchanged to mouse.
Construct Hu9338 Hu9006 5882 C8-H241 36C4 5D5 F206P 127 96 31 103 104 100D208G 75 69 39 95 71 100H209Y 94 90 49 84 43 100P210S 100 79 18 105 89 100K223Q 198 112 96 19 127 100human MET 149 134 96 77 115 100 Antibody binding is expressed as the percentage of 5D5 binding. Grey cells indicate less than 50% antibody binding compared to 5D5.
PCT/IB2015/002110 WO 2016/042412 Table 8. Summary of the binding epitopes identified for tested MET antibodies using cell surface expressed mutated MET constructs.
Antibody Domain SEMA Blade Chimeric Fragment Residues (AA) Linear epitope Contact Residues Epitope Bin HGF blocking Hu9338 SEMA-a 2 AA 84-122 BL 2 AA 99-113 N.D. Bin 8 Yes C8-H241 SEMA-a 2-3 AA 163-224BL 2 AA 119-1BL 3 AA 209-233BL 3 K223 Bin 7 Yes 36C4 SEMA-a 2-3 AA 84 - 224 BL 2: 129-143 BL 3 H209 Bin 8 Yes Hu9006 SEMA-a 3 AA 163-224 N.D. N.D. Bin 6 Yes 5882 SEMA-a 3 AA 163-224 N.D.BL 3 F206, D208, H209, P210Bin 9 No 5D5 SEMA-a 5 AA 313-371 N.D. N.D. Bin 4 Yes 224G11 IPT1 N.A. AA 562-652 N.D. N.D. Bin 1 Yes Abbreviations: AA: Amino Acid sequence. N.A: Not applicable. N.D: Not determined. BL: Blade.
Example 8: Affinity Measurements for Chimeric and Humanized Anti-MET Antibodies id="p-301" id="p-301" id="p-301" id="p-301" id="p-301"
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[0301]This example demonstrates that the humanized variants of anti-MET antibodies 9006 and 9338 have affinities comparable to their chimeric counterparts, indicating that the humanized antibodies have the full functional activity of the chimeric antibodies.Furthermore, the humanized anti-MET antibodies show comparable binding to both human and cynomolgus MET ECD.
Methods id="p-302" id="p-302" id="p-302" id="p-302" id="p-302"
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[0302]Kinetic binding analysis of the purified humanized and chimeric 9006 and 93variants was performed on an Octet QK384 Bio-Layer Interferometry (BLI) biosensor (Fortebio, USA) or an XPR-36 surface plasmon resonance (SPR) biosensor (Bio-Rad, USA). [0303]His tagged human or cynomolgus MET ECD antigens were purchased from Sinobiological, China. Binding kinetics were measured under monovalent antigen conditions by immobilizing anti-MET antibodies and keeping the monovalent MET antigen in solution as described previously (Canziani et al., Anal Biochem 325(2):301-307 (2004). The lowest possible anti-MET antibody density was applied to prevent non-specific binding and mass transport limitation. For measuring antibody kinetics on the Octet system, antibodies at a concentration of 1.5 pg/ml were captured on anti-human Fc sensors (Fortebio, USA), and tested for binding to human MET ECD antigen (100 nM) serially diluted two-fold seven times.
PCT/IB2015/002110 WO 2016/042412 Measurements were conducted with a plate rotation speed of 1000 rpm and sensors were regenerated and reused by brief 5 second alternations between exposure to 10 mM Glycine:HCI buffer (pH 1.5) or PBS buffer containing 1% BSA and 0.001% Tween 20 three times. For the Surface Plasmon Resonance experiments conducted on the Bio-Rad XPR-instrument, anti-MET antibodies were adjusted to a concentration of 0.25-0.5 pg/ml and captured on anti-human IgG Fc surfaces generated by immobilizing a monoclonal anti- human Fc antibody (Biacore, Denmark). Anti-MET antibodies were tested for binding to human or cynomolgus MET ECD in a 2-fold concentration range from 25 nM to 1.56 nM followed by regeneration of the surfaces with 3 M MgCI2 regeneration buffer (Biacore, Denmark). The recorded binding responses were fitted to a simple Langmuir 1:1 binding model for calculation of the on-rate (kon or ka), off-rate (koff or kd) and affinity (KD) constants using double referencing.
Results id="p-304" id="p-304" id="p-304" id="p-304" id="p-304"
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[0304]The kinetic measurements using the Octet biosensor showed that the humanized variant of 9006 with 3 back mutations (Hu9006) and the humanized variant of 93(Hu9338) with no back mutations have slightly improved affinity for the human MET antigen compared to the chimeric parent antibodies (Table 9).
Table 9. Binding kinetics of chimeric and humanized MET antibodies to human MET ECD as measured by Bio-Layer Interferometry (BLI).
Antibody MET ECD kon (M-1 s-1) kon Error koff (s-1) koff Error KD (M) 9006 human 5.4E+04±4.4E+02 1.2E-04±2.2E-06 2.2E-09 hu9006 human 6.2E+04±6.8E+02 7.6E-05±3.1E-06 1.2E-09 9338 human 1.9E+05±2.7E+03 1.1E-04±4.0E-06 6.0E-10 hu9338 human 1.0E+05±2.0E+03 2.7E-05±3.6E-06 2.6E-10 id="p-305" id="p-305" id="p-305" id="p-305" id="p-305"
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[0305]The kinetic measurements using the Bio-Rad XPR36 SPR instrument showed that Hu9006 and Hu9338 recognize both human and cynomolgus MET ECD with affinities in the pM range (Table 10).
Table 10. Binding kinetics of humanized MET antibodies to human or cynomolgus MET ECD as measured by Surface Plasmon Resonance (SPR).
Antibody MET ECD kon (M-1 s-1) kon Error koff (s-1) koff Error KD (M) PCT/IB2015/002110 WO 2016/042412 hu9006 human 1.9E+05±1.2E+03 1.1E-05±2.3E-07 5.5E-11 hu9006 cynomolgus 1.8E+05±1.5E+03 1.6E-05±2.9E-07 8.6E-11 hu9338 human 4.7E+05±1.9E+02 6.3E-06±3.7E-07 1.4E-11 hu9338 cynomolgus 7.4E+05±2.2E+03 5.4E-05±3.6E-07 7.4E-11 Example 9: Degradation of MET With Anti-MET Antibodies id="p-306" id="p-306" id="p-306" id="p-306" id="p-306"
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[0306]This example demonstrates that the anti-MET antibodies 9006 and 9338 induce degradation of MET, alone and in combination. The combination of the two antibodies induces more efficient degradation of the MET receptor than either antibody alone.
Methods id="p-307" id="p-307" id="p-307" id="p-307" id="p-307"
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[0307]To investigate the level of MET receptor degradation induced by individual anti- MET antibodies 9006 and 9338, the mixture of 9006 and 9338, and the C8-H241 analogue (see Table 4), Western Blot or Simple Western analysis was performed on whole cell lysates of SNU5, EBC1 and MKN45 cells treated with antibody for 24 or 48 hours. In brief, cells were grown in T-75 culture flasks, and when 50% confluent the culture media were removed, the cells were washed and treated with a 20 pg/ml total antibody concentration of either C8- H241, 9006, 9338, 9338+9006, or a negative control antibody (human lgG1 against a non- mammalian target) for 24 or 48 hours in a humidified incubator at 37°C. Whole cell lysates were prepared using standard RIPA buffer. The total protein concentration was determined using a BCA assay and 1-10 pg protein analyzed by the Simple Western automated immunoassay on a Sally instrument (ProteinSimple) or by Western Blot analysis using primary detection antibodies against MET. An antibody against 3-actin was used as loading control for the Western blot analysis.
Results id="p-308" id="p-308" id="p-308" id="p-308" id="p-308"
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[0308]The results from the Western Blot investigation (Figure 3) show that treatment with the individual antibodies (especially 9006) induces some degradation of MET in all cell lines tested. However, the anti-MET antibody mixture 9338+9006 induces enhanced MET receptor degradation compared with the individual antibodies (9006 or 9338) across all cell lines tested. The cellular MET receptor level after 24 hours or treatment with 9006+9338 or C8-H241 was compared by Simple Western analysis in the three cell lines SNU5, EBC and MKN45. Results shown in Figure 4 demonstrate enhanced MET degradation after treatment with 9006+9338 in all three cell lines.
Example 10: Inhibition of MET Phosphorylation and Downstream Signaling with Anti- MET Antibodies PCT/IB2015/002110 WO 2016/042412 id="p-309" id="p-309" id="p-309" id="p-309" id="p-309"
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[0309]This example demonstrates that the anti-MET antibodies 9006 and 9338 have differential and cell line-dependent effects on MET phosphorylation and downstream signaling (as determined by levels of pERK2 and pAKT). The anti-MET antibody mixture 9006+9338 induces efficient inhibition of MET phosphorylation and downstream signaling.
Methods id="p-310" id="p-310" id="p-310" id="p-310" id="p-310"
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[0310]To investigate the level of inhibition of MET phosphorylation and downstream signaling induced by anti-MET antibodies 9006 and 9338 and the anti-MET antibody mixture 9006+9338, Simple Western analysis was performed on whole cell lysates of MKN45 and EBC-1 cells treated with antibody for 24 hours. Cells were grown in 6-well plates. When 50% confluent, the culture media was removed, and the cells were washed in 1xPBS and treated with 20 pg/ml total antibody concentration (9006, 9338, 9006+9338, or the negative control antibody Synagis®) for 24 hours in a humidified incubator at 37°C. Whole cell lysates were prepared using standard RIPA buffer. The total protein concentration was determined using a BCA assay, and approximately 1 mg/ml protein analyzed by Simple Western analysis using a Sally instrument (automated size-based immunoassay system, ProteinSimple) and by using primary antibodies against phosphorylated MET (Tyr1234/12and Tyr1349), phosphorylated ERK2 (pERK2), and phosphorylated AKT (pAKT). An antibody against 3-actin was used as loading control (data not shown).
Results id="p-311" id="p-311" id="p-311" id="p-311" id="p-311"
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[0311]The results from the Simple Western analysis of phosphorylation levels of MET (Figure 5) and ERK2 and AKT (Figure 6) show that treatment with 9006 or 9338 alone induces differential and cell line-dependent effects on phosphorylation in the cell lines tested. The anti-MET antibody mixture 9006+9338, however, induces efficient inhibition of MET phosphorylation and downstream signaling compared to treatment with monoclonal anti- MET antibody 9006 or 9338 in both MKN45 and EBC-1 cells.
Example 11: Anti-Proliferative Effect of Chimeric Anti-MET Antibodies in Primary Endothelial Cells id="p-312" id="p-312" id="p-312" id="p-312" id="p-312"
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[0312]Human umbilical vein endothelial cells (HUVEC) are primary endothelial cells suitable for evaluating biological effects in a sensitive vascular model. The anti-MET antibodies 9006 and 9338 and the antibody mixture 9006+9338 are shown to be able to inhibit the growth of HUVECs, both in the absence and presence of the MET ligand HGF.
Materials and Methods id="p-313" id="p-313" id="p-313" id="p-313" id="p-313"
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[0313]Dermal fibroblast cells were thawed and seeded in seeding medium in 96-well plates. After sedimentation of the fibroblasts at room temperature, a vial of GFP labeled- PCT/IB2015/002110 WO 2016/042412 HUVECs was thawed. The HUVECs, resuspended in seeding medium, were added on top of the fibroblast suspension and incubated overnight in an Incucyte instrument (Essen Bioscience) at 37°C and 5% C02. After overnight incubation, medium from the co-cultured cells was removed and replaced with growth medium for an additional 24 hours. The following day, assay medium was prepared, and different ligand/antibody mixtures were combined and mixed into the assay medium. The growth medium was removed and replaced with the assay medium containing the different combination of antibodies/ligands. The medium was exchanged with fresh assay medium containing antibody/ligand mixtures every two to three days. Pictures of GFP-HUVECs were recorded every four hours. Several cell parameters, including cell number, cell network length, and number of network branching points were analyzed using Incucyte software.
Results id="p-314" id="p-314" id="p-314" id="p-314" id="p-314"
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[0314]Figures 7-11 show the efficacy of antibodies 9006 and 9338 antibodies in specifically inhibiting primary endothelial cell proliferation, in contrast to an unrelated antibody control that does not show any inhibitory effect. The antibody mixture 9006+93demonstrates superior inhibition of HUVEC proliferation, particularly when HGF is present in the medium.
Example 12: In Vitro Comparison of Chimeric and Humanized Anti-MET Antibodies id="p-315" id="p-315" id="p-315" id="p-315" id="p-315"
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[0315]This example describes in vitro comparison of chimeric 9006, chimeric 9338 and chimeric 9338+9006 with the humanized variants i.e. humanized 9006 (Hu9006), humanized 9338 (Hu9338) and humanized 9338+9006 (Hu9338+Hu9006). The monoclonal antibodies and the mixture were evaluated for their ability to inhibit the growth of several cancer cell lines: Okajima, EBC1, MKN45, HCC827R1_cet#3, HCC827R1_cet#1 and Katoll.
Methods id="p-316" id="p-316" id="p-316" id="p-316" id="p-316"
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[0316]The 9006, 9338, 9338+9006 (1:1 mixture of the two components), Hu9006, Hu93and Hu9338+Hu9006 (1:1 mixture of the two components) along with the negative control antibody (Synagis®) were diluted to a final total antibody concentration of 100 pg/ml in RPMI 1640 Glutamax media supplemented with 2% FBS and 1% P/S, yielding a final concentration of 25 pg/ml in wells containing the highest antibody concentration. A twofold serial dilution of the antibodies was then performed, giving up to 17 different concentrations. Relevant numbers of cells (Okajima: 1000 cells/well, EBC1: 750 cells/well, MKN45: 5cells/well, HCC827R1_cet#3: 500 cells/well, HCC827R1_cet#1: 500 cells/well; Katoll: 7cells/well) were added to the experimental wells in a 384 well plate, and incubated with antibodies for 4 days in a humidified incubator at 37°C. WST-1 reagent was subsequently added to the plates and incubated for one hour at 37°C. The absorbance was measured at PCT/IB2015/002110 WO 2016/042412 450 nm and 620 nm (reference wavelength) using an ELISA reader. The absorbance at 6nM were subtracted from the absorbance at 450 nM, and the amount of metabolically active cells (MAC) was calculated as a percentage of the untreated control as described in Example 2.
Results id="p-317" id="p-317" id="p-317" id="p-317" id="p-317"
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[0317]Figures 12 and 13 depict the viability results from titrations of chimeric and humanized 9006 and 9338 antibodies and the chimeric and humanized 9006+9338 antibody mixture on the cell lines HCC827R1_cet#3 (12A), HCC827R1_cet#1 (12B), MKN45 (12C), EBC-1 (13A), Katoll (13B), and Okajima (13C). It is evident from the graphs that Hu9006, Hu9338, and Hu9338+Hu9006 have an anti-proliferative effect comparable to that of their chimeric counterparts. Example 13: In Vitro Comparison of Humanized 9338+9006 and 13-MET+28-MET id="p-318" id="p-318" id="p-318" id="p-318" id="p-318"
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[0318]This example describes the in vitro testing of humanized 9338+90(Hu9338+Hu9006), 13-MET, 28-MET and 13-MET+28-MET (see Table 4). The monoclonal antibodies and the mixtures were evaluated for their ability to inhibit the growth of four cancer cell lines: EBC1, MKN45, SNU5 and Katoll.
Methods id="p-319" id="p-319" id="p-319" id="p-319" id="p-319"
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[0319]Antibodies Hu9338+Hu9006 (1:1 mixture of the two components), 13-MET, 28-MET and 13-MET+28-MET (1:1 mixture of the two components) along with the negative control antibody (Synagis®) were tested for anti-metabolic effect in EBC1 (500 cells/well), MKN(750 cells/well), SNU5 (750 cells/well) and Katoll (750 cells/well) as described above.
Results id="p-320" id="p-320" id="p-320" id="p-320" id="p-320"
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[0320]The viability results from titrations of Hu9338+Hu9006, 13-MET, 28-MET and 13- MET+28-MET antibodies on the cell lines EBC1, MKN45, SNU5 and Katoll are shown in Figure 14. It is evident that the anti-MET antibodies have different levels of efficacy and potency depending on the cell line tested. However, the combination of Hu9338 and Hu90demonstrate superior inhibition of metabolic activity compared with 13-MET, 28-MET and 13-MET+28-MET across all cell lines tested.
Example 14: In Vivo Efficacy of the Chimeric 9006+9338 Antibody Mixture in a Human EBC-1 Tumor Xenograft Model id="p-321" id="p-321" id="p-321" id="p-321" id="p-321"
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[0321]This example demonstrates in vivo efficacy of the 9006+9338 antibody mixture in xenografts of the human MET-amplified non-small cell lung cancer cell line EBC-1.
Methods PCT/IB2015/002110 WO 2016/042412 id="p-322" id="p-322" id="p-322" id="p-322" id="p-322"
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[0322] 5 x 106 EBC-1 cells were inoculated subcutaneously into the flanks of 8-9 week oldfemale athymic nude mice. Tumors were measured three times weekly by caliper in two dimensions and tumor volume in mm3 was calculated according to the formula: (width)2 x length x 0.5. At an average tumor size of 120mm3, the mice were randomized and treatment was initiated. The mice were treated three times weekly for a total of ten treatments by intraperitoneal injection of vehicle buffer (10 mM sodium citrate, 150 mM sodium chloride, pH 6.0), monoclonal antibody 9006, monoclonal antibody 9338, ora 1:1 mixture of monoclonal antibodies 9006+9338, followed by an observation period. All antibody treatments were administered at 50 mg/kg total antibody concentration. Thus, 9006- and 9338-treated animals were dosed with 50 mg/kg of 9006 or 9338, respectively, whereas animals treated with 9006+9338 were dosed with a mixture containing 25 mg/kg of each antibody.
Results id="p-323" id="p-323" id="p-323" id="p-323" id="p-323"
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[0323]On day 10 post-inoculation, at an average tumor size of 120 mm3, the mice were randomized into four groups of eight animals and treatment was initiated. As shown in Figure 15, treatment with monoclonal antibody 9338 did not affect tumor growth in animals compared to the vehicle control. In contrast, treatment with 9006 resulted in tumor growth delay, whereas treatment with 9006+9338 induced growth stabilization during treatment and was superior to all other treatments in this model. Studies of the groups treated with vehicle or with 9006 or 9338 alone were closed during the treatment period due to tumor outgrowth or tumor related ulcerations, whereas animals in the 9006+9338 group completed treatment and observation for two to three weeks after the end of treatment.
Example 15: In Vivo Efficacy of Increasing Doses of the Chimeric 9006+9338 Antibody Mixture in a Human EBC-1 Tumor Xenograft Model id="p-324" id="p-324" id="p-324" id="p-324" id="p-324"
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[0324]This example demonstrates in vivo efficacy of increasing doses of the 9006+93antibody mixture in xenografts of the human MET-amplified non-small cell lung cancer cell line EBC-1.
Methods id="p-325" id="p-325" id="p-325" id="p-325" id="p-325"
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[0325] 5 x 106 EBC-1 cells were inoculated subcutaneously into the flanks of 8-9 week oldfemale athymic nude mice. Tumors were measured three times weekly by caliper in two dimensions and tumor volume in mm3 was calculated according to the formula: (width)2 x length x 0.5. At an average tumor size of 150 mm3, the mice were randomized and treatment was initiated. The mice were treated three times weekly for a total of ten treatments by intraperitoneal injection of vehicle buffer (10 mM sodium citrate, 150 mM sodium chloride, pH 6.0) or a 1:1 mixture of monoclonal antibodies 9006+9338, followed by PCT/IB2015/002110 WO 2016/042412 an observation period. The 1:1 mixture of 9006+9338 was administered at 50, 25, 5 or mg/kg total antibody concentration per injected dose.
Results id="p-326" id="p-326" id="p-326" id="p-326" id="p-326"
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[0326]On day 11 post-inoculation, at an average tumor size of 120 mm3, the mice were randomized into five groups of ten animals and treatment was initiated. As shown in Figure 16, tumor growth was not affected in animals treated with the lowest concentration of 9006+9338 (1 mg/kg) compared to vehicle control treated animals. Treatment with 5 mg/kg 9006+9338 resulted in tumor growth delay at later time points, whereas treatment with 25 or mg/kg 9006+9338 induced comparable levels of potent tumor inhibition with growth stabilization.
Example 16: In Vivo Efficacy of the Chimeric 9006+9338 Antibody Mixture in a Human MKN-45 Tumor Xenograft Model id="p-327" id="p-327" id="p-327" id="p-327" id="p-327"
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[0327]This example demonstrates in vivo efficacy of the 9006+9338 antibody mixture in xenografts of the human MET-amplified gastric cancer cell line MKN-45.
Methods id="p-328" id="p-328" id="p-328" id="p-328" id="p-328"
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[0328] 5 x 106 MKN-45 cells were inoculated subcutaneously into the flanks of 8-9 weekold female athymic nude mice. Tumors were measured three times weekly by caliper in two dimensions and tumor volume in mm3 was calculated according to the formula: (width)2 x length x 0.5. At an average tumor size of 80 mm3, the mice were randomized and treatment was initiated. The mice were treated three times weekly for a total of ten treatments by intraperitoneal injection of vehicle buffer (10 mM sodium citrate, 150 mM sodium chloride, pH 6.0), monoclonal antibody 9006, monoclonal antibody 9338, ora 1:1 mixture of monoclonal antibodies 9006+9338, followed by an observation period. All antibody treatments were administered at 50 mg/kg total antibody concentration. Thus, 9006- and 9338-treated animals were dosed with 50 mg/kg of 9006 or 9338, respectively, whereas animals treated with 9006+9338 were dosed with a mixture containing 25 mg/kg of each antibody.
Results id="p-329" id="p-329" id="p-329" id="p-329" id="p-329"
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[0329]On day 10 post-inoculation, at an average tumor size of 80 mm3, the mice were randomized into four groups of eight animals and treatment was initiated. As shown in Figure 17, tumor growth was slightly inhibited in animals treated with monoclonal antibody 9006 or 9338 alone compared to vehicle control treated animals. In contrast, treatment with 9006+9338 induced growth stabilization during treatment and was superior to all other PCT/IB2015/002110 WO 2016/042412 treatments in this model. Studies of groups treated with vehicle or 9006 alone were closed during the treatment period due to tumor outgrowth or tumor related ulcerations, whereas the animals in the 9338 and 9006+9338 groups completed treatment. The 9006+9338 group was observed for 2 weeks after the end of treatment, and growth stabilization was retained during most of this period.
Example 17: In Vivo Efficacy of the Chimeric 9006+9338 Antibody Mixture in a Human SNU5 Tumor Xenograft Model id="p-330" id="p-330" id="p-330" id="p-330" id="p-330"
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[0330]This example demonstrates in vivo efficacy of the 9006+9338 antibody mixture in xenografts of the human MET-amplified gastric cancer cell line SNU5.
Methods id="p-331" id="p-331" id="p-331" id="p-331" id="p-331"
id="p-331"
[0331] 1 x 107 SNU5 cells were inoculated subcutaneously into the flanks of 8-9 week oldfemale athymic nude mice. Tumors were measured three times weekly by caliper in two dimensions and tumor volume in mm3 was calculated according to the formula: (width)2 x length x 0.5. At an average tumor size of 165 mm3׳, the mice were randomized and treatment was initiated. The mice were treated three times weekly for a total of ten treatments by intraperitoneal injection of vehicle buffer (10 mM sodium citrate, 150 mM sodium chloride, pH 6.0), monoclonal antibody 9006, monoclonal antibody 9338, or a 1:mixture of monoclonal antibodies 9006+9338, followed by an observation period. All antibody treatments were administered at 50 mg/kg total antibody concentration. Thus,9006- and 9338-treated animals were dosed with 50 mg/kg of 9006 or 9338, respectively, whereas animals treated with 9006+9338 were dosed with a mixture containing 25 mg/kg of each antibody.
Results id="p-332" id="p-332" id="p-332" id="p-332" id="p-332"
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[0332]On day 15 post-inoculation, at an average tumor size of 165 mm3, the mice were randomized into four groups of eight animals and treatment was initiated. As shown in Figure 18, tumor regression was observed in animals treated with monoclonal antibody 90or 9338 or with the 9006+9338 antibody mixture compared to vehicle control treated animals. Treatment with 9006 or 9006+9338 was superior to treatment with 9338, and tumor regression was retained for more than 50 days after the end of treatment in the 9006- and 9006+9338-treated groups.
Example 18: In Vivo Efficacy of the Chimeric 9006+9338 Antibody Mixture in a Human Hepatocellular Carcinoma Patient-Derived Xenograft Model id="p-333" id="p-333" id="p-333" id="p-333" id="p-333"
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[0333]This example demonstrates in vivo efficacy of the 9006+9338 antibody mixture in a patient-derived xenograft model (L11037) of human hepatocellular carcinoma (HCC).
PCT/IB2015/002110 WO 2016/042412 Methods id="p-334" id="p-334" id="p-334" id="p-334" id="p-334"
id="p-334"
[0334]The tumor source for model L11037 is derived from a liver cancer patient tumor which was then maintained subcutaneously in nude mice. Tumors were minced into 3 mmfragments, and one fragment was implanted subcutaneously at one front flank in each mouse. The animals were randomized into treatment groups when the tumor reached 2mm3 mean volume. The mice were treated three times weekly for a total of ten treatments by intraperitoneal injection of vehicle buffer (10 mM sodium citrate, 150 mM sodium chloride, pH 6.0) or a 1:1 mixture of monoclonal antibodies 9006+9338, followed by an observation period. All antibody treatments were administered at 50 mg/kg total antibody concentration. Thus, animals treated with 9006+9338 were dosed with a mixture containing 25 mg/kg of each antibody.
Results id="p-335" id="p-335" id="p-335" id="p-335" id="p-335"
id="p-335"
[0335] On day 21 post-inoculation, at an average tumor size of 220 mm3, the mice were randomized into two groups of four animals and treatment was initiated. As shown in Figure 19, tumor growth inhibition was observed in animals treated with the 9006+9338 antibody mixture compared to vehicle control treated animals.
Example 19: In Vivo Comparison of Chimeric and Humanized Antibody Mixtures in a Human EBC-1 Tumor Xenograft Model id="p-336" id="p-336" id="p-336" id="p-336" id="p-336"
id="p-336"
[0336]In this example the in vivo efficacies of the chimeric 9006+9338 and the humanized Hu9006+Hu9338 antibody mixtures are compared in xenografts of the human MET amplified non-small cell lung cancer cell line EBC-1.
Methods id="p-337" id="p-337" id="p-337" id="p-337" id="p-337"
id="p-337"
[0337] 5x106 EBC-1 cells were inoculated subcutaneously into the flank of 8-9 week oldfemale athymic nude mice. Tumors were measured three times weekly by caliper in two dimensions and tumor volume in mm3 was calculated according to the formula: (width)2 x length x 0.5. On day 20 post cell inoculation at an average tumor size of -130 mm3, the mice were randomized into three groups of 10 animals and treatment was initiated. The mice were treated three times weekly with a total of ten intraperitoneal injections of vehicle buffer, a 1:mixture of chimeric 9006+9338 or humanized 9006+9338 (Hu9006+Hu9338) followed by an observation period. All antibody treatments were dosed at 50 mg/kg total antibody concentration. Thus, animals treated with 9006+9338 and Hu9006+Hu9338 were dosed with a mixture containing 25 mg/kg of each antibody.
Results PCT/IB2015/002110 WO 2016/042412 id="p-338" id="p-338" id="p-338" id="p-338" id="p-338"
id="p-338"
[0338]As shown in Figure 20, tumor regression was observed in animals treated with both 9006+9338 and Hu9006+Hu9338 as compared to vehicle control treated animals. The tumor inhibitory effect of Hu9006+Hu9338 and 9006+9338 appeared highly similar.
Example 20: In Vivo Comparison of Chimeric and Humanized Antibody Mixtures in a Human OE33 Tumor Xenograft Model id="p-339" id="p-339" id="p-339" id="p-339" id="p-339"
id="p-339"
[0339]In this example, the in vivo efficacies of the chimeric 9006+9338 and the humanized Hu9006+Hu9338 antibody mixtures are compared in xenografts of the human MET amplified esophagogastric cancer cell line OE33.
Methods id="p-340" id="p-340" id="p-340" id="p-340" id="p-340"
id="p-340"
[0340]OE33 tumors were serially transplanted from previously established tumors.Tumors had been passaged eight times at the time of study. Tumor fragments measuring ~mm3 were transplanted subcutaneously into the flank of 8-9 week old female athymic nude mice. Tumors were measured three times weekly by caliper in two dimensions and tumor volume in mm3 was calculated according to the formula: (width)2 x length x 0.5. On day post-tumor inoculation an average tumor size of 200 mm3, the mice were randomized into three groups of seven animals and treatment was initiated. The mice were treated three times weekly with a total of ten treatments by intraperitoneal injection of vehicle buffer, a 1:mixture of chimeric 9006+9338 or humanized 9006+9338 (Hu9006+Hu9338) followed by an observation period. All antibody treatments were dosed at 30 mg/kg total antibody concentration. Thus, animals treated with 9006+9338 and Hu9006+Hu9338 were dosed with a mixture containing 15 mg/kg of each antibody.
Results id="p-341" id="p-341" id="p-341" id="p-341" id="p-341"
id="p-341"
[0341]As shown in Figure 21, tumor regression was observed in animals treated with both 9006+9338 and Hu9006+Hu9338 compared to vehicle control treated animals and the growth curves are highly similar.
Example 21: In vivo Comparison of the monoclonal antibody C8-H241 and the Hu9006+Hu9338 antibody mixture in human tumor xenograft models id="p-342" id="p-342" id="p-342" id="p-342" id="p-342"
id="p-342"
[0342]In this example, the in vivo efficacies of the Hu9006+Hu9338 antibody mixture and the comparator monoclonal antibody C8-H241 (see Table 4) are compared in xenografts of the human MET amplified non-small cell lung cancer cell line EBC-1 and the human MET amplified gastric cancer cell line Hs746T, which also harbors a MET exon 14 deletion.
Methods PCT/IB2015/002110 WO 2016/042412 id="p-343" id="p-343" id="p-343" id="p-343" id="p-343"
id="p-343"
[0343] 5x106 EBC-1 cells or 3.7x106 Hs746T cells were inoculated subcutaneously into theflank of female athymic mice. Tumors were measured three times weekly by caliper in two dimensions and tumor volume in mm3 was calculated according to the formula: (width)2 x length x 0.5. At an average tumor size of 140 mm3 for EBC-1 and 120 mm3 for Hs746T the mice were randomized and treatment was initiated. [0344]Treatment schedule for EBC-1: The mice were treated three times weekly with a total often intraperitoneal injections of vehicle buffer, monoclonal antibody C8-H241, ora 1:mixture of monoclonal antibodies Hu9006+Hu9338 followed by an observation period. Afterdays of observation, remaining mice in the C8-H241 group were re-treated with Hu9006+Hu9338 three times weekly until study termination on day 139 after tumor cell inoculation. [0345]Treatment schedule for Hs746T: The mice were treated three times weekly with a total often intraperitoneal injections of vehicle buffer, monoclonal antibody C8-H241, monoclonal antibody Hu9006, monoclonal antibody Hu9338 or a 1:1 mixture of monoclonal antibodies Hu9006+Hu9338. After a one week observation period all remaining mice in the Hu9006, Hu9338 and C8-H241 groups were treated with a single dose of Hu9006+Hu93and observed for 9 days. [0346]All antibody treatments were dosed at 50 mg/kg total antibody concentration. Thus, C8-H241, Hu9006 and Hu9338 treated animals were dosed with 50 mg/kg antibody whereas animals treated with Hu9006+Hu9338 were dosed with a mixture containing 25 mg/kg of each antibody.
Results id="p-347" id="p-347" id="p-347" id="p-347" id="p-347"
id="p-347"
[0347]EBC-1: On day 15 post-inoculation at an average tumor size of 140 mm3 the mice were randomized into three groups of ten animals and treatment was initiated. As shown in Figure 22, a limited response was observed in mice treated with C8-H241 compared to vehicle control treated animals. In contrast, treatment with Hu9006+Hu9338 induced tumor regression. 21 days after the last dose, at an average tumor volume of 500 mm3, the remaining mice in the C8-H241 treated group were re-treated with Hu9006+Hu9338. Figurealso shows that the mice responded with tumor regression upon the secondary treatment. [0348] Hs746T: On day 35 post-inoculation at an average tumor size of 120 mm3 the micewere randomized into five groups of eight animals and treatment was initiated. As shown in Figure 23, a limited initial inhibitory response was observed in mice treated with C8-H241, Hu9006 or Hu9338 compared to vehicle control treated animals, but approximately halfway through the treatment period, the tumors started to re-grow. In contrast, treatment with Hu9006+Hu9338 induced tumor regression and complete tumor eradication in all eight mice treated. Nine days after the last dose, the remaining mice in the C8-H241, Hu9006 and PCT/IB2015/002110 WO 2016/042412 Hu9338 treated groups were re-treated with a single dose of Hu9006+Hu9338. Figure also shows that the mice responded with tumor regression upon the secondary treatment.
Example 22: In Vivo Comparison of the monoclonal antibody C8-H241 and the Hu9006+Hu9338 antibody mixture in four human patient derived xenograft models id="p-349" id="p-349" id="p-349" id="p-349" id="p-349"
id="p-349"
[0349]In this example, the in vivo efficacy of the Hu9006+Hu9338 antibody mixture and the comparator monoclonal antibody C8-H241 (see Table 4) were compared in four human MET amplified non-small cell lung cancer (NSCLC) patient derived xenograft models.
Methods id="p-350" id="p-350" id="p-350" id="p-350" id="p-350"
id="p-350"
[0350]Each mouse was inoculated subcutaneously at the flank with primary NSCLC tissue fragments from model LXFA0526, LU0858, LU1901 or LU2503 (2-3 mm in diameter) for tumor development. Tumors were measured two times weekly by caliper in two dimensions and tumor volume in mm3 was calculated according to the formula: (width)2 x length x 0.5. [0351]When average tumor size reached 100-200 mm3, mice were randomly assigned into three groups (n=5 to 8 mice per group) and treatment was initiated. Mice were treated three times weekly for a total of ten intraperitoneal injections with either C8-H2monoclonal antibody, Hu9006+Hu9338 antibody mixture (single monoclonal antibodies mixed at equal ratio) or vehicle buffer control followed by an observation period of up to three weeks. [0352]All antibody treatments were dosed at 50 mg/kg total antibody concentration. Thus, C8-H241 treated animals were dosed with 50 mg/kg antibody whereas animals treated with Hu9006+Hu9338 were dosed with a mixture containing 25 mg/kg of each antibody.
Results id="p-353" id="p-353" id="p-353" id="p-353" id="p-353"
id="p-353"
[0353]As shown in Figure 24, varying responses were observed in the four models upon C8-H241 treatment. In contrast, treatment with Hu9006+Hu9338 induced tumor regression in all 4 models with superior efficacy and/or delayed time to progression compared to C8- H241. C8-H241 was previously reported to be highly efficacious in a different MET amplified primary MET amplified xenograft NSCLC model (LXFA-1647) (Liu et al. Clin Cancer Res. 20:6059-6070 (2014)).
Example 23: In Vivo Comparison of balanced and skewed ratio compositions of the Hu9006+Hu9338 antibody mixture in a human tumor xenograft model id="p-354" id="p-354" id="p-354" id="p-354" id="p-354"
id="p-354"
[0354]In this example, the in vivo efficacy of mixtures consisting of different ratios of the two antibodies Hu9006 and Hu9338 was compared in xenografts of the human MET amplified non-small cell lung cancer cell line EBC-1.
PCT/IB2015/002110 WO 2016/042412 Methods id="p-355" id="p-355" id="p-355" id="p-355" id="p-355"
id="p-355"
[0355] 5x106 EBC-1 cells were inoculated subcutaneously into the flank of 8-9 week oldfemale athymic nude mice. Tumors were measured three times weekly by caliper in two dimensions and tumor volume in mm3 was calculated according to the formula: (width)2 x length x 0.5. On day 13 post-inoculation at an average tumor size of 150 mm3 the mice were randomized into three groups of ten animals and treatment was initiated. The mice were treated three times weekly with a total of ten intraperitoneal injections of vehicle buffer, 1:1, 2:1 or 1:2 skewed antibody ratio mixtures of monoclonal antibodies Hu9006+Hu93followed by an observation period. Antibody treatments were dosed at either 50 mg/kg or, for the skewed antibody ratio mixtures, 10mg/kg total antibody concentration as follows: 1:ratio dosed animals were dosed with a mixture containing 25 mg/kg of each antibody. 1:ratio dosed animals were either dosed with a mixture containing 3 mg/kg Hu9006 and mg/kg Hu9338 for a total dosing of 10 mg/kg or with a mixture containing 17 mg/kg Hu90and 33 mg/kg Hu9338 for a total dosing of 50 mg/kg. Analogously, 2:1 ratio dosed animals were either dosed with a mixture containing 7 mg/kg Hu9006 and 3 mg/kg Hu9338 for a total dosing of 10 mg/kg or with a mixture containing 33 mg/kg Hu9006 and 17 mg/kg Hu9338 for a total dosing of 50 mg/kg.
Results id="p-356" id="p-356" id="p-356" id="p-356" id="p-356"
id="p-356"
[0356]As shown in Figure 25, treatment with Hu9006+Hu9338 at both balanced and skewed ratios and at both doses induced tumor regression. The level of tumor regression appeared similar for all tested antibody treatments indicating that Hu9006+Hu9338 yields a robust and consistent tumor growth inhibition at both balanced and skewed single antibody compositions.
Table 11: SEQ ID NO Chart SEQ ID SequenceNOhuman MET isoform 1 amino acid sequencehuman MET isoform 2 amino acid sequencechicken MET amino acid sequencemurine MET amino acid sequencechimeric 9006 heavy chain variable domain nucleic acid sequencechimeric 9006 heavy chain variable domain amino acid sequencechimeric 9006 light chain variable domain nucleic acid sequencechimeric 9006 light chain variable domain amino acid sequencechimeric 9338 heavy chain variable domain nucleic acid sequencechimeric 9338 heavy chain variable domain amino acid sequencechimeric 9338 light chain variable domain nucleic acid sequencechimeric 9338 light chain variable domain amino acid sequencehumanized 9006 heavy chain variable domain nucleic acid sequencehumanized 9006 heavy chain variable domain amino acid sequence PCT/IB2015/002110 WO 2016/042412 humanized 9006 light chain variable domain nucleic acid sequencehumanized 9006 light chain variable domain amino acid sequencehumanized 9338 heavy chain variable domain nucleic acid sequencehumanized 9338 heavy chain variable domain amino acid sequencehumanized 9338 light chain variable domain nucleic acid sequencehumanized 9338 light chain variable domain amino acid sequence9006 heavy chain CDR1 amino acid sequence9006 heavy chain CDR2 amino acid sequence9006 heavy chain CDR3 amino acid sequence9006 light chain CDR1 amino acid sequence9006 light chain CDR2 amino acid sequence9006 light chain CDR3 amino acid sequence9338 heavy chain CDR1 amino acid sequence9338 heavy chain CDR2 amino acid sequence9338 heavy chain CDR3 amino acid sequence9338 light chain CDR1 amino acid sequence9338 light chain CDR2 amino acid sequence9338 light chain CDR3 amino acid sequencehumanized 9006 light chain amino acid sequencehumanized 9006 heavy chain amino acid sequencehumanized 9338 light chain amino acid sequencehumanized 9338 heavy chain amino acid sequence PCT/IB2015/002110 WO 2016/042412 List of Sequences SEQ ID NO: 1 (human MET isoform 1 amino acid sequence):MKAPAVLAPGILVLLFTLVQRSNGECKEALAKSEMNVNMKYQLPNFTAETPIQNVIL HEHHIFLGATNYIYVLNEEDLQKVAEYKTGPVLEHPDCFPCQDCSSKANLSGGVWKD NINMALWDTYYDDQLISCGSVNRGTCQRHVFPHNHTADIQSEVHCIFSPQIEEPSQ CPDCWSALGAKVLSSVKDRFINFFVGNTINSSYFPDHPLHSISVRRLKETKDGFMF LTDQSYIDVLPEFRDSYPIKYVHAFESNNFIYFLTVQRETLDAQTFHTRIIRFCSIN SGLHSYMEMPLECILTEKRKKRSTKKEVFNILQAAYVSKPGAQLARQIGASLNDDIL FGVFAQSKPDSAEPMDRSAMCAFPIKYVNDFFNKIVNKNNVRCLQHFYGPNHEHCFN RTLLRNSSGCEARRDEYRTEFTTALQRVDLFMGQFSEVLLTSISTFIKGDLTIANLG TSEGRFMQVWSRSGPSTPHVNFLLDSHPVSPEVIVEHTLNQNGYTLVITGKKITKI PLNGLGCRHFQSCSQCLSAPPFVQCGWCHDKCVRSEECLSGTWTQQICLPAIYKVFP NSAPLEGGTRLTICGWDFGFRRNNKFDLKKTRVLLGNESCTLTLSESTMNTLKCTVG PAMNKHFNMSIIISNGHGTTQYSTFSYVDPVITSIS PKYG PMAGGTLLTLTGNYLNS GNSRHISIGGKTCTLKSVSNSILECYTPAQTISTEFAVKLKIDLANRETSIFSYRED PIVYEIHPTKSFISGGSTITGVGKNLNSVSVPRMVINVHEAGRNFTVACQHRSNSEI ICCTTPSLQQLNLQLPLKTKAFFMLDGILSKYFDLIYVHNPVFKPFEKPVMISMGNE NVLEIKGNDIDPEAVKGEVLKVGNKSCENIHLHSEAVLCTVPNDLLKLNSELNIEWK QAISSTVLGKVIVQPDQNFTGLIAGWSISTALLLLLGFFLWLKKRKQIKDLGSELV RYDARVHTPHLDRLVSARSVSPTTEMVSNESVDYRATFPEDQFPNSSQNGSCRQVQY PLTDMSPILTSGDSDISSPLLQNTVHIDLSALNPELVQAVQHWIGPSSLIVHFNEV IGRGHFGCVYHGTLLDNDGKKIHCAVKSLNRITDIGEVSQFLTEGIIMKDFSHPNVL SLLGICLRSEGSPLWLPYMKHGDLRNFIRNETHNPTVKDLIGFGLQVAKGMKYLAS KKFVHRDLAARNCMLDEKFTVKVADFGLARDMYDKEYYSVHNKTGAKLPVKWMALES LQTQKFTTKSDVWS FGVLLWELMTRGAP PY PDVNTFDITVYLLQGRRLLQ PEYC PD P LYEVMLKCWHPKAEMRPSFSELVSRISAIFSTFIGEHYVHVNATYVNVKCVAPYPSL LSSEDNADDEVDTRPASFWETS SEQ ID NO: 2 (human MET isoform 2 amino acid sequence):MKAPAVLAPGILVLLFTLVQRSNGECKEALAKSEMNVNMKYQLPNFTAETPIQNVIL HEHHIFLGATNYIYVLNEEDLQKVAEYKTGPVLEHPDCFPCQDCSSKANLSGGVWKD NINMALWDTYYDDQLISCGSVNRGTCQRHVFPHNHTADIQSEVHCIFSPQIEEPSQ CPDCWSALGAKVLSSVKDRFINFFVGNTINSSYFPDHPLHSISVRRLKETKDGFMF LTDQSYIDVLPEFRDSYPIKYVHAFESNNFIYFLTVQRETLDAQTFHTRIIRFCSIN SGLHSYMEMPLECILTEKRKKRSTKKEVFNILQAAYVSKPGAQLARQIGASLNDDIL FGVFAQSKPDSAEPMDRSAMCAFPIKYVNDFFNKIVNKNNVRCLQHFYGPNHEHCFN RTLLRNSSGCEARRDEYRTEFTTALQRVDLFMGQFSEVLLTSISTFIKGDLTIANLG TSEGRFMQWVSRSGPSTPHVNFLLDSHPVSPEVIVEHTLNQNGYTLVITGKKITKI PLNGLGCRHFQSCSQCLSAPPFVQCGWCHDKCVRSEECLSGTWTQQICLPAIYKVFP NSAPLEGGTRLTICGWDFGFRRNNKFDLKKTRVLLGNESCTLTLSESTMNTLKCTVG PAMNKHFNMSIIISNGHGTTQYSTFSYVDPVITSIS PKYG PMAGGTLLTLTGNYLNS GNSRHISIGGKTCTLKSVSNSILECYTPAQTISTEFAVKLKIDLANRETSIFSYRED PIVYEIHPTKSFISTWWKEPLNIVSFLFCFASGGSTITGVGKNLNSVSVPRMVINVH EAGRNFTVACQHRSNSEIICCTTPSLQQLNLQLPLKTKAFFMLDGILSKYFDLIYVH NPVFKPFEKPVMISMGNENVLEIKGNDIDPEAVKGEVLKVGNKSCENIHLHSEAVLC TVPNDLLKLNSELNIEWKQAISSTVLGKVIVQPDQNFTGLIAGWSISTALLLLLGF FLWLKKRKQIKDLGSELVRYDARVHTPHLDRLVSARSVSPTTEMVSNESVDYRATFP EDQFPNSSQNGSCRQVQYPLTDMSPILTSGDSDISSPLLQNTVHIDLSALNPELVQA VQHWIGPSSLIVHFNEVIGRGHFGCVYHGTLLDNDGKKIHCAVKSLNRITDIGEVS QFLTEG11MKDFSHPNVLSLLGICLRSEGS PLWLPYMKHGDLRNFIRNETHNPTVK DLIGFGLQVAKGMKYLASKKFVHRDLAARNCMLDEKFTVKVADFGLARDMYDKEYYS VHNKTGAKLPVKWMALESLQTQKFTTKSDVWSFGVLLWELMTRGAPPYPDVNTFDIT VYLLQGRRLLQPEYCPDPLYEVMLKCWHPKAEMRPSFSELVSRISAIFSTFIGEHYV HVNATYVNVKCVAPYPSLLSSEDNADDEVDTRPASFWETS SEQ ID NO: 3 (chicken MET amino acid sequence):MKPVTAYPSGIILFLFALLQRSHGQCKEAAKKSEMNLNVKYDLPNFITETPIQNWL YKHHVYIGAVNKIYVLNETLQNISVYKTGPILESPGCAPCEDCKDKANLSNSVWKDN VNMALLLETYYDDQLIS CGSVSGGVCHRHIIP PDNPADIE S EVHCMYSPQVDGEADN PCT/IB2015/002110 WO 2016/042412 CPDCWSTLGTKVLVTEKDRFVNFFVGNTMTSAFQPPHVLHSISVRRLKETQDGFEF LTDQSYIDILPQFRDSYPIKYVHAFEHDHFVYFLTVQRESLDSQTFHTRIIRFCTLD SEMRSYMEMPLECIFTEKRRKRSIRKEVFNILQAAYVSKPGAALAHEMGLGLIDDIL YGVFAQTNQIPQEPTNRSAVCAVSVRTINEFFNKIVDKQNMKCLQHFYGKDSKYCLN RAFSRNASYCRAQDDEYRLEVTTPLQRVDLFMGQFNNILLTSISVFTKGNLTIANLG TS EGRFMQIWSRS EPTAPHVS FQLDSHAVS PQVWEQSAAADGYTLWTGKKITKV PLNGPGCHHFQSCSQCLLAPAFMRCGWCGQQCLRAPECNGGTWTQETCLPRVYEILP SSAPLEGGTKLTLCGWDFGFSKNNRFELRNTWHIGGQICALEAKSSNKNKLECTAP AAKNASFNISSSVSVGHGKTLFNTFSYVNPIITSISPTYGPKSGGTLLTIAGKYLNS GKSRRIFVGEKPCSLKSTSESSVECYTPAQRIPQEYRVRVGIDGAIRDAKGYFTYRE DPWLKIHPAKSFLSGGSTITAQGINLNSVCFPRMVITVPKLGMNFSVACSHRSSSE IICCTTPSLKAFNLQPPFVTKVFFIFDGVSSLYFDFDYVNNPVFKHFEKPVLISRSN PNVLEIKGNHIDSEAVKGEVLKVGNKSCENLLLQSETILCTVPSDLLKSNSELNIEW KQEVLSTVIGKVLIRQDQNFTGLIAGWSTSVLIYIFLVFFLWRRKKKQIKDLGSDL VRYDGRVHTPHLDRLVSARSVSPTTEMVSSESVDYRSTFLEDQFPSMSQNGSCRPAQ YPHSDLSPILSSGDSDLASPLLQTNVHIDISALNPDLVKEVQHWIGADSLMVHFSE VIGRGHFGCVSHGTLLDNDGRKIHCAVKSLNRITDLEEVAQFLKEGIIMKDFTHPNV LSLLGI CLPNEGSPLWLPYMKHGDLRNFIRNETHNPTVKDLIGFGLQVAKGMKYLA SKKFVHRDLAARNCMLDEKFTVKVADFGLARDVYDKEYYSVHNKTGAKLPVKWMALE SLQTQKFTTKSDVWSFGVLLWELMTRGAPPYPDVNSFDITVYLLQGRRLLQPEYCPD PLYEVMLKCWHPKPEMRPAFSELVSKISTIFSTFIGEHYVHVNATYVNVKCVAPYPS LLSSQDNTDMDVDT SEQ ID NO: 4 (murine MET amino acid sequence):MKAPTVLAPGILVLLLSLVQRSHGECKEALVKSEMNVNMKYQLPNFTAETPIQNWL HGHHIYLGATNYIYVLNDKDLQKVSEFKTGPVLEHPDCLPCRDCSSKANSSGGVWKD NINMALLVDTYYDDQLISCGSVNRGTCQRHVLPPDNSADIQSEVHCMFSPEEESGQC PDCWSALGAKVLLSEKDRFINFFVGNTINSSYPPGYSLHSISVRRLKETQDGFKFL TDQSYIDVLPEFQDSYPIKYIHAFESNHFIYFLTVQKETLDAQTFHTRIIRFCSVDS GLHSYMEMPLECILTEKRRKRSTREEVFNILQAAYVSKPGANLAKQIGASPSDDILF GVFAQSKPDSAEPVNRSAVCAFPIKYVNDFFNKIVNKNNVRCLQHFYGPNHEHCFNR TLLRNSSGCEARSDEYRTEFTTALQRVDLFMGRLNQVLLTSISTFIKGDLTIANLGT SEGRFMQWLSRTAHLTPHVNFLLDSHPVSPEVIVEHPSNQNGYTLWTGKKITKIP LNGLGCGHFQSCSQCLSAPYFIQCGWCHNQCVRFDECPSGTWTQEICLPAVYKVFPT SAPLEGGTVLTICGWDFGFRKNNKFDLRKTKVLLGNESCTLTLSESTTNTLKCTVGP AMSEHFNVSVIISNSRETTQYSAFSYVDPVITSISPRYGPQAGGTLLTLTGKYLNSG NSRHISIGGKTCTLKSVSDSILECYTPAQTTSDEFPVKLKIDLANRETSSFSYREDP WYEIHPTKSFISGGSTITGIGKTLNSVSLPKLVIDVHEVGVNYTVACQHRSNSEII CCTTPSLKQLGLQLPLKTKAFFLLDGILSKHFDLTYVHNPVFEPFEKPVMISIGNEN WEIKGNNIDPEAVKGEVLKVGNQSCESLHWHSGAVLCTVPSDLLKLNSELNIEWKQ AVSSTVLGKVIVQPDQNFAGLIIGAVSISVWLLLSGLFLWMRKRKHKDLGSELVRY DARVHTPHLDRLVSARSVSPTTEMVSNESVDYRATFPEDQFPNSSQNGACRQVQYPL TDLSPILTSGDSDISSPLLQNTVHIDLSALNPELVQAVQHWIGPSSLIVHFNEVIG RGHFGCVYHGTLLDNDGKKIHCAVKSLNRITDIEEVSQFLTEGIIMKDFSHPNVLSL LGICLRSEGSPLWLPYMKHGDLRNFIRNETHNPTVKDLIGFGLQVAKGMKYLASKK FVHRDLAARNCMLDEKFTVKVADFGLARDMYDKEYYSVHNKTGAKLPVKWMALESLQ TQKFTTKSDVWS FGVLLWELMTRGAP PY PDVNTFDITIYLLQGRRLLQ PEYC PDALY EVMLKCWHPKAEMRPSFSELVSRISSIFSTFIGEHYVHVNATYVNVKCVAPYPSLLP SQDNIDGEGNT SEQ ID NO: 5 (chimeric 9006 heavy chain variable domain nucleic acid sequence):CAGATCCATTTGGGGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCACAAACTTTAGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATGTTGATGACTTGAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACATGGCTACATATTTCTGTGCAAGGAAAGGGATTGCGAGGGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCGAGT SEQ ID NO: 6 (chimeric 9006 heavy chain variable domain amino acid sequence): PCT/IB2015/002110 WO 2016/042412 QIHLGQSGPELKKPGETVKISCKASGYTFTNFRMNWVKQAPGKGLKWMGWINTYTGEPTYVDDLKGRFAFSLETSASTAYLQINNLKNEDMATYFCARKGIARAMDYWGQGTSVTVSS SEQ ID NO: 7 (chimeric 9006 light chain variable domain nucleic acid sequence):AACATTGTGATGACACAGTCTCCATCCTCCCTGAGTGTGTCAGCAGGAGAGATGGTC ACTATGAGTTGTAAGTCCAGTCAGAGTCTGTTAGACAGTGGAAATCAAAAGAACTAC TTGGCCTGGTACCAGCAGAAACCAGGGCAGCCTCCTCAACTTTTGATCTTCGGGGCA TCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACCGAT TTCACTCTTACCGTCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAG AATGAT CATAGTTATC CGTACACGTT CGGAGGGGGGAC CAAG CTGGAAATAAAA SEQ ID NO: 8 (chimeric 9006 light chain variable domain amino acid sequence):NIVMTQSPSSLSVSAGEMVTMSCKSSQSLLDSGNQKNYLAWYQQKPGQPPQLLIFGASTRESGVPDRFTGSGSGTDFTLTVSSVQAEDLAVYYCQNDHSYPYTFGGGTKLEIK SEQ ID NO: 9 (chimeric 9338 heavy chain variable domain nucleic acid sequence):CAGGTCCAACTGCAACAGCCTGGGGCTGAACTGGCAAAACCTGGGGCCTCAGTGAGGATGTCCTGCAAGGCTTCTGGCTACACCTTTACTAGTTACTGGATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATTAATCCTAGCAGTGGTCATATTGAGAACAATCAGAAGTTCAAGGACAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATTTGAGGACTCTGCAGTCTATTACTGTGCAAGAGGACGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCGAGT SEQ ID NO: 10 (chimeric 9338 heavy chain variable domain amino acid sequence):QVQLQQPGAELAKPGASVRMSCKASGYTFTSYWMHWVKQRPGQGLEWIGYINPSSGHIENNQKFKDKATLTADKSSSTAYMQLSSLTFEDSAVYYCARGRFAYWGQGTLVTVSS SEQ ID NO: 11 (chimeric 9338 light chain variable domain nucleic acid sequence):GATATTGTGATGACCCAGTCTCCAGCAATCATGTCTGCATCTCCTGGGGAGAAGGTCACCTTGACCTGCAGTGCCAGCTCAAGTGTAAGTTCCGGCTACTTGTACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACTCTGGATTTATAGCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAGTCAACAGCATGGAGGCTGAAGATGCTGCCTCTTATTTCTGCCATCAGTGGAGTAGTTACCCATTCACGTTCGGCTCGGGGACCAAGCTGGAGCTGAAA SEQ ID NO: 12 (chimeric 9338 light chain variable domain amino acid sequence):DIVMTQSPAIMSASPGEKVTLTCSASSSVSSGYLYWYQQKPGSSPKLWIYSTSNLAS GVPARFSGSGSGTSYSLTVNSMEAEDAASYFCHQWSSYPFTFGSGTKLELK SEQ ID NO: 13 (humanized 9006 heavy chain variable domain nucleic acid sequence):CAGGTGCAGCTGGTGCAGTCTGGATCCGAGCTGAAGAAACCTGGCGCCTCCGTGAAGGTGTCCTGCAAGGCTTCCGGCTACACCTTTACCAACTTCCGGATGAACTGGGTCAAGCAGGCCCCAGGCCAGGGCCTGAAATGGATGGGCTGGATCAACACCTACACCGGCGAGCCCACCTACGTGGACGACCTGAAGGGCAGATTCGTGTTCTCCCTGGACACCTCCGTGTCCACCGCCTACCTGCAGATCTCCAGCCTGAAGGCCGAGGATACCGCCGTGTACTACTGCGCCCGGAAGGGAATCGCCAGAGCCATGGATTATTGGGGCCAGGGCACCACCGTGACAGTCTCGAGT SEQ ID NO: 14 (humanized 9006 heavy chain variable domain amino acid sequence):QVQLVQSGSELKKPGASVKVSCKASGYTFTNFRMNWVKQAPGQGLKWMGWINTYTGEPTYVDDLKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARKGIARAMDYWGQGTTVTVSS SEQ ID NO: 15 (humanized 9006 light chain variable domain nucleic acid sequence): 100 PCT/IB2015/002110 WO 2016/042412 GACATCGTGATGACCCAGTCCCCCGACTCTCTGGCCGTGTCTCTGGGCGAGAGAGCCACCATCAACTGCAAGTCCTCCCAGTCCCTGCTGGACTCCGGCAACCAGAAGAACTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGCCTCCCAAGCTGCTGATCTTTGGCGCCTCCACCCGGGAATCTGGCGTGCCCGATAGATTCTCCGGCTCCGGCTCTGGCACCGACTTTACCCTGACCATCAGCTCCCTGCAGGCCGAGGATGTGGCCGTGTACTACTGCCAGAACGACCACTCCTACCCCTACACCTTCGGCCAGGGCACCAAGCTGGAAATCAAG SEQ ID NO: 16 (humanized 9006 light chain variable domain amino acid sequence):DIVMTQSPDSLAVSLGERATINCKSSQSLLDSGNQKNYLAWYQQKPGQPPKLLIFGASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDHSYPYTFGQGTKLEIK SEQ ID NO: 17 (humanized 9338 heavy chain variable domain nucleic acid sequence):CAGGTGCAGCTGGTGCAGTCTGGCGCTGAAGTGAAGAAACCCGGCTCCTCCGTGAAGGTGTCCTGCAAGGCCTCCGGCTACACCTTTACCAGCTACTGGATGCACTGGGTGCGACAGGCCCCTGGACAGGGCCTGGAATGGATGGGCTACATCAACCCCTCCAGCGGCCACATCGAGAACAACCAGAAATTCAAGGACCGCGTGACCATCACCGCCGACAAGTCCACCTCCACCGCCTACATGGAACTGTCCTCCCTGCGGAGCGAGGACACCGCCGTGTACTACTGTGCCAGAGGCAGATTCGCCTACTGGGGCCAGGGCACCCTCGTGACAGTCTCGAGT SEQ ID NO: 18 (humanized 9338 heavy chain variable domain amino acid sequence):QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGYINPSSGHIENNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARGRFAYWGQGTLVTVSS SEQ ID NO: 19 (humanized 9338 light chain variable domain nucleic acid sequence):GAGATCGTGCTGACCCAGTCTCCTGCCACCCTGTCTCTGAGCCCTGGCGAGAGAGCTACCCTGTCCTGCTCCGCCTCCTCCTCTGTGTCCTCCGGCTACCTGTACTGGTATCAGCAGAAGCCCGGCCAGGCCCCTCGGCTGCTGATCTACTCTACCTCCAACCTGGCCTCCGGCATCCCTGCCAGATTCTCCGGCTCTGGCTCTGGCACCGACTTTACCCTGACCATCTCCAGCCTGGAACCCGAGGACTTCGCCGTGTACTACTGCCACCAGTGGTCCAGCTACCCCTTCACCTTTGGCTCCGGCACCAAGCTGGAAATCAAG SEQ ID NO: 20 (humanized 9338 light chain variable domain amino acid sequence):EIVLTQS PATLSLS PGERATLSCSAS SSVS SGYLYWYQQKPGQAPRLLIYSTSNLAS GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCHQWSSYPFTFGSGTKLEIK SEQ ID NO: 21 (9006 heavy chain CDR1 amino acid sequence):GYTFTNFR SEQ ID NO: 22 (9006 heavy chain CDR2 amino acid sequence):INTYTGEP SEQ ID NO: 23 (9006 heavy chain CDR3 amino acid sequence):ARKGIARAMDY SEQ ID NO: 24 (9006 light chain CDR1 amino acid sequence):QSLLDSGNQKNY SEQ ID NO: 25 (9006 light chain CDR2 amino acid sequence):GAS SEQ ID NO: 26 (9006 light chain CDR3 amino acid sequence):QNDHSYPYT 101
Claims (36)
1. An antibody composition comprising a first anti-MET antibody or an antigen-bindingportion thereof and a second anti-MET antibody or an antigen-binding portion thereof, wherein:the H-CDR1-3 and L-CDR1-3 of said first anti-MET antibody comprise the amino acid sequences of SEQ ID NOs: 21, 22, 23, 24, 25, and 26, respectively, andthe H-CDR1-3 and L-CDR1-3 of said second anti-MET antibody comprise the amino acid sequences of SEQ ID NOs: 27, 28, 29, 30, 31, and 32, respectively.
2. The antibody composition of claim 1, wherein:the VH and VL of said first anti-MET antibody comprise the amino acid sequences of SEQ ID NOs: 6 and 8, respectively, andthe VH and VL of said second anti-MET antibody comprise the amino acid sequences of SEQ ID NOs: 10 and 12, respectively.
3. The antibody composition of claim 1, wherein:the VH and VL of said first anti-MET antibody comprise the amino acid sequences of SEQ ID NOs: 14 and 16, respectively, andthe VH and VL of said second anti-MET antibody comprise the amino acid sequences of SEQ ID NOs: 18 and 20, respectively.
4. The antibody composition of any one of claims 1-3, wherein the first anti-MET antibody,the second anti-MET antibody, or both are of isotype IgG.
5. The antibody composition of claim 4, wherein the first anti-MET antibody, the secondanti-MET antibody, or both are of isotype subclass IgG1.
6. An antibody composition comprising a first anti-MET antibody and a second anti-METantibody, wherein: 103 250902/4 the HC and LC of said first anti-MET antibody comprise the amino acidsequences of SEQ ID NOs: 34 and 33, respectively, andthe HC and LC of said second anti-MET antibody comprise the amino acid sequences of SEQ ID NO: 36 and 35, respectively.
7. The antibody composition of any one of claims 1-6, wherein each antibody in saidcomposition has at least one property selected from the group consisting of:a) does not bind to mouse or chicken MET;b) binds to an epitope of human MET comprising residues that are present on theSEMA domain;c) induces degradation of MET;d) binds to human MET with a KD of 1 x 10-9 M or less;e) inhibits growth in vitro of at least one cell line selected from SNU5, EBC1,MKN45, KatoII, OE33, and Okajima;f) inhibits MET phosphorylation;g) inhibits MET downstream signaling;h) inhibits primary endothelial cell proliferation in the presence or absence of HGF;andi) inhibits tumor growth in vivo.
8. The antibody composition of any one of claims 1-7, wherein said composition has at leastone property selected from the group consisting of:a) induces degradation of MET;b) inhibits growth in vitro of at least one cell line selected from SNU5, EBC1,MKN45, KatoII, OE33, and Okajima;c) inhibits MET phosphorylation;d) inhibits MET downstream signaling;e) inhibits primary endothelial cell proliferation in the presence or absence of HGF;andf) inhibits tumor growth in vivo. 104 250902/4
9. A pharmaceutical composition comprising the antibody composition of any one of claims1-8 and a pharmaceutically acceptable excipient.
10. An anti-MET antibody or an antigen-binding portion thereof, wherein the H-CDR1-3 and L-CDR1-3 of said antibody comprise the amino acid sequences of SEQ ID NOs: 21, 22, 23, 24, 25, and 26, respectively.
11. The anti-MET antibody or antigen-binding portion of claim 10, wherein said antibody has a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 6 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 8.
12. The anti-MET antibody or antigen-binding portion of claim 10, wherein said antibody has a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 14 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 16.
13. An anti-MET antibody or an antigen-binding portion thereof, wherein the H-CDR1-3 and L-CDR1-3 of said antibody comprise the amino acid sequences of SEQ ID NOs: 27, 28, 29, 30, 31, and 32, respectively.
14. The anti-MET antibody or antigen-binding portion of claim 13, wherein said antibody has a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 12.
15. The anti-MET antibody or antigen-binding portion of claim 13, wherein said antibody has a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 20. 105 250902/4
16. An anti-MET antibody, wherein the heavy chain (HC) of said antibody comprises the amino acid sequence of SEQ ID NO: 34 and the light chain (LC) of said antibody comprises the amino acid sequence of SEQ ID NO: 33.
17. An anti-MET antibody, wherein the heavy chain (HC) of said antibody comprises the amino acid sequence of SEQ ID NO: 36 and the light chain (LC) of said antibody comprises the amino acid sequence of SEQ ID NO: 35.
18. The antibody or antigen-binding portion of any one of claims 10-17, wherein the antibody is of isotype IgG.
19. The antibody or antigen-binding portion of claim 18, wherein the antibody is of isotype subclass IgG1.
20. The anti-MET antibody or antigen-binding portion of any one of claims 10-19, wherein said antibody has at least one property selected from the group consisting of:a) does not bind to mouse or chicken MET;b) binds to an epitope of human MET comprising residues that are present on theSEMA domain;c) induces degradation of MET;d) binds to human MET with a KD of 1 x 10-9 M or less;e) inhibits growth in vitro of at least one cell line selected from SNU5, EBC1,MKN45, KatoII, OE33, and Okajima;f) inhibits MET phosphorylation;g) inhibits MET downstream signaling;h) inhibits primary endothelial cell proliferation in the presence or absence of HGF;andi) inhibits tumor growth in vivo.
21. A pharmaceutical composition comprising the anti-MET antibody or antigen-binding portion of any one of claims 10-20 and a pharmaceutically acceptable excipient. 106 250902/4
22. An isolated nucleic acid molecule comprising a nucleotide sequence that encodes the heavy chain or an antigen-binding portion thereof, and a nucleotide sequence that encodes the light chain or an antigen-binding portion thereof, of the anti-MET antibody of any one of claims 10-20.
23. The isolated nucleic acid molecule of claim 22, comprising the nucleotide sequence ofSEQ ID NO: 5, 7, 9, 11, 13, 15, 17, or 19.
24. A vector comprising the isolated nucleic acid molecule of claim 22 or 23, wherein said vector further comprises an expression control sequence.
25. A host cell comprising a nucleotide sequence that encodes the heavy chain or an antigenbinding portion thereof, and a nucleotide sequence that encodes the light chain or an antigen-binding portion thereof, of the anti-MET antibody of any one of claims 10-20.
26. A method for producing the antibody or antigen-binding portion of any one of claims 1020, comprising providing a host cell according to claim 25, cultivating said host cell under conditions suitable for expression of the antibody or portion, and isolating the resulting antibody or portion.
27. A method for producing the antibody composition of any one of claims 1-8, comprising providing a first host cell capable of expressing the first anti-MET antibody or antigenbinding portion and a second host cell capable of expressing the second anti-MET antibody or antigen-binding portion, cultivating said first and second host cells under conditions suitable for expression of the antibodies or portions, and isolating the resulting antibodies or portions, optionally wherein the first and second host cells are cultured in a single bioreactor.
28. A bispecific binding molecule comprising a first antigen-binding domain comprising the amino acid sequences of SEQ ID NOs: 21, 22, 23, 24, 25, and 26, respectively; and a 107 250902/4 second antigen-binding domain comprising the amino acid sequences of SEQ ID NOs: 27, 28, 29, 30, 31, and 32, respectively.
29. The antibody composition of any one of claims 1-8 or the pharmaceutical composition of claim 9 for use in treating a patient with a MET-mediated disorder.
30. The anti-MET antibody or antigen-binding portion of any one of claims 10-20, the pharmaceutical composition of claim 21, or the bispecific binding molecule of claim for use in treating a patient with a MET-mediated disorder.
31. The antibody composition of any one of claims 1-8 or the pharmaceutical composition of claim 9 for use in treating cancer in a patient.
32. The anti-MET antibody or antigen-binding portion of any one of claims 10-20, the pharmaceutical composition of claim 21, or the bispecific binding molecule of claim for use in treating cancer in a patient.
33. The antibody composition, pharmaceutical composition, anti-MET antibody or antigenbinding portion, or bispecific binding molecule for use of claim 31 or 32, wherein the cancer is dependent on MET activation.
34. The antibody composition, pharmaceutical composition, anti-MET antibody or antigenbinding portion, or bispecific binding molecule for use of claim 31 or 32, wherein the cancer is non-small cell lung cancer, gastric cancer, hepatocellular carcinoma, esophageal cancer, colorectal cancer, kidney papillary cell cancer, glioblastoma, renal cell carcinoma, prostate cancer, or adrenocortical carcinoma.
35. The antibody composition, pharmaceutical composition, anti-MET antibody or antigenbinding portion, or bispecific binding molecule for use of any one of claims 29-34, wherein said use further comprises administering a chemotherapeutic agent, an anti- 108 250902/4 neoplastic agent, an anti-angiogenic agent, a tyrosine kinase inhibitor, or a MET pathway inhibitor.
36. The antibody composition, pharmaceutical composition, anti-MET antibody or antigenbinding portion, or bispecific binding molecule for use of any one of claims 29-35, wherein the patient is a human. For the Applicants REINHOLD COHN AND PARTNERS By: 109
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- 2015-09-15 BR BR112017005202A patent/BR112017005202A2/en active Search and Examination
- 2015-09-15 CN CN201580059801.8A patent/CN107001471B/en active Active
- 2015-09-15 KR KR1020177010306A patent/KR102200274B1/en active IP Right Grant
- 2015-09-15 ES ES15804578T patent/ES2748295T3/en active Active
- 2015-09-15 WO PCT/IB2015/002110 patent/WO2016042412A1/en active Application Filing
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WO2016042412A1 (en) | 2016-03-24 |
EP3194444B1 (en) | 2019-07-10 |
CA2961323A1 (en) | 2016-03-24 |
KR20170054517A (en) | 2017-05-17 |
MX2017003211A (en) | 2017-06-06 |
BR112017005202A2 (en) | 2017-12-12 |
EP3194444A1 (en) | 2017-07-26 |
CN107001471A (en) | 2017-08-01 |
IL250902A0 (en) | 2017-04-30 |
KR102200274B1 (en) | 2021-01-08 |
US20180327500A1 (en) | 2018-11-15 |
CN107001471B (en) | 2022-01-18 |
AU2015316522A1 (en) | 2017-03-23 |
ES2748295T3 (en) | 2020-03-16 |
US10450376B2 (en) | 2019-10-22 |
AU2015316522B2 (en) | 2021-01-21 |
JP6927875B2 (en) | 2021-09-01 |
CA2961323C (en) | 2021-11-30 |
JP2017534259A (en) | 2017-11-24 |
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